Engineering single-gene-controlled staygreen potential into plants

ABSTRACT

The enzymes of the ACC synthase family are used in producing ethylene. Nucleotide and polypeptide sequences of ACC synthases are provided along with knockout plant cells having inhibition in expression and/or activity in an ACC synthase and knockout plants displaying a staygreen phenotype, a male sterility phenotype, or an inhibition in ethylene production. Methods for modulating staygreen potential in plants, methods for modulating sterility in plants, and methods for inhibiting ethylene production in plants are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional utility patent applicationclaiming priority to and benefit of the following prior provisionalpatent application: U.S. Ser. No. 60/480,861, filed Jun. 23, 2003,entitled “Engineering single-gene-controlled staygreen potential intoplants” by Gallie et al., which is incorporated herein by reference inits entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.95-35304-4657 from USDA/CREES and support under Grant MCB-0076434-002from the National Science Foundation. The government may have certainrights to this invention.

FIELD OF THE INVENTION

This invention relates to modulating staygreen potential in plants,inhibiting ethylene production in plants, and modulating sterility inplants. The invention also provides knockout plant cells, e.g., wherethe knockout plant cells are disrupted in ACC synthase expression and/oractivity, or knockout plants, e.g., which display a staygreen phenotypeor a male sterility phenotype. Nucleic acid sequences and amino acidsequences encoding various ACC synthases are also included.

BACKGROUND OF THE INVENTION

Stay-green is a term used to describe a plant phenotype, e.g., wherebyleaf senescence (most easily distinguished by yellowing of the leafassociated with chlorophyll degradation) is delayed compared to astandard reference. See, Thomas H and Howarth C J (2000) “Five ways tostay green” Journal of Experimental Botany 51:329-337. In sorghum,several stay-green genotypes have been identified which exhibit a delayin leaf senescence during grain filling and maturation. See, Duncan R R,et al. (1981) “Descriptive comparison of senescent and non-senescentsorghum genotypes.” Agronomy Journal 73:849-853. Moreover, underconditions of limited water availability, which normally hastens leafsenescence (see, e.g., Rosenow D T, and Clark L E (1981) Droughttolerance in sorghum. In: Loden H D, Wilkinson D, eds. Proceedings ofthe 36th annual corn and sorghum industry research conference, 18-31),these genotypes retain more green leaf area and continue to fill grainnormally (see, e.g., McBee G G, Waskom R M, Miller F R, Creelman R A(1983) Effect of senescence and non-senescence on carbohydrates insorghum during late kernel maturity states. Crop Science 23:372-377;Rosenow D T, Quisenberry J E, Wendt C W, Clark L E (1983)Drought-tolerant sorghum and cotton germplasm. Agricultural WaterManagement 7:207-222; and, Borrell A K, Douglas A C L (1996) Maintaininggreen leaf area in grain sorghum increases yield in a water-limitedenvironment. In: Foale M A, Henzell R G, Kneipp J F, eds. Proceedings ofthe third Australian sorghum conference. Melbourne: Australian Instituteof Agricultural Science, Occasional Publication No. 93). The stay-greenphenotype has also been used as a selection criterion for thedevelopment of improved varieties of corn, particularly with regard tothe development of drought-tolerance. See, e.g., Russell W A (1991)Genetic improvement of maize yields. Advances in Agronomy 46: 245-298;and, Bruce et al. (2002), “Molecular and physiological approaches tomaize improvement for drought tolerance” Journal of Experimental Botany,53 (366): 13-25.

Five fundamentally distinct types of stay-green have been described,which are Types A, B, C, D and E (see e.g., Thomas H, Smart C M (1993)Crops that stay green. Annals of Applied Biology 123:193-219; and,Thomas and Howarth, supra). In Type A stay-green, initiation of thesenescence program is delayed, but then proceeds at a normal rate. InType B stay-green, while initiation of the senescence program isunchanged, the progression is comparatively slower. In Type Cstay-green, chlorophyll is retained even though senescence (asdetermined through measurements of physiological function such asphotosynthetic capacity) proceeds at a normal rate. Type D stay-green ismore artificial in that killing of the leaf (i.e. by freezing, boilingor drying) prevents initiation of the senescence program, therebystopping the degradation of chlorophyll. In Type E stay-green, initiallevels of chlorophyll are higher, while initiation and progression ofleaf senescence are unchanged, thereby giving the illusion of arelatively slower progression rate. Type A and B are functionalstay-greens, as photosynthetic capacity is maintained along withchlorophyll content, and these are the types associated with increasedyield and drought tolerance in sorghum. Despite the potential importanceof this trait, in particular the benefits associated with increasingyield and drought tolerance, very little progress has been made inunderstanding the biochemical, physiological or molecular basis forgenetically determined stay-green (Thomas and Howarth, supra).

This invention solves these and other problems. The invention relates tothe identification of ACC synthase genes associated with staygreenpotential phenotype in plants and modulation of staygreen potentialand/or ethylene production. Polypeptides encoded by these genes, methodsfor modulating staygreen potential in plants, methods for inhibitingethylene production in plants, methods for modulating sterility inplants, and knockout plant cells and plants, as well as other features,will become apparent upon review of the following materials.

SUMMARY OF THE INVENTION

This invention provides methods and compositions for modulatingstaygreen potential and sterility in plants and modulating (e.g.,inhibiting) ethylene synthesis and/or production in plants. Thisinvention also relates to ACC synthase nucleic acid sequences in plants,exemplified by, e.g., SEQ ID NO:1 through SEQ ID NO:6 and SEQ ID NO:10,and a set of polypeptide sequences, e.g., SEQ ID NO:7 through SEQ IDNO:9 and SEQ ID NO:11, which can modulate these activities.

In a first aspect, the invention provides for an isolated or recombinantknockout plant cell comprising at least one disruption in at least oneendogenous ACC synthase gene (e.g., a nucleic acid sequence, orcomplement thereof, comprising, e.g., at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 99.5% or more sequenceidentity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), or SEQ ID NO:3(gACS7)). The disruption inhibits expression or activity of at least oneACC synthase protein compared to a corresponding control plant celllacking the disruption. In one embodiment, the at least one endogenousACC synthase gene comprises two or more endogenous ACC synthase genes(e.g., any two or more of ACS2, ACS6, and ACS7, e.g., ACS2 and ACS6).Similarly, in another embodiment, the at least one endogenous ACCsynthase gene comprises three or more endogenous ACC synthase genes. Incertain embodiments, the disruption results in reduced ethyleneproduction by the knockout plant cell as compared to the control plantcell.

In one embodiment, the at least one disruption in the knockout plantcell is produced by introducing at least one polynucleotide sequencecomprising an ACC synthase nucleic acid sequence, or subsequencethereof, into a plant cell, such that the at least one polynucleotidesequence is linked to a promoter in a sense or antisense orientation,and where the at least one polynucleotide sequence comprises, e.g., atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about99.5% or more sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2(gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6),SEQ ID NO:6 (cACS7), or SEQ ID NO:10 (CCRA178R) or a subsequencethereof, or a complement thereof. In another embodiment, the disruptionis introduced into the plant cell by introducing at least onepolynucleotide sequence comprising one or more subsequences of an ACCsynthase nucleic acid sequence configured for RNA silencing orinterference.

In another embodiment, the disruption comprises insertion of one or moretransposons, where the one or more transposons are in the at least oneendogenous ACC synthase gene. In yet another embodiment, the disruptioncomprises one or more point mutations in the at least one endogenous ACCsynthase gene. The disruption can be a homozygous disruption in the atleast one ACC synthase gene. Alternatively, the disruption is aheterozygous disruption in the at least one ACC synthase gene. Incertain embodiments, when more than one ACC synthase gene is involved,there is more than one disruption, which can include homozygousdisruptions, heterozygous disruptions or a combination of homozygousdisruptions and heterozygous disruptions.

In certain embodiments, a plant cell of the invention is from a dicot ormonocot. In one aspect, the plant cell is in a hybrid plant comprising astaygreen potential phenotype. In another aspect, the plant cell is in aplant comprising a sterility phenotype, e.g., a male sterilityphenotype. Plants regenerated from the plant cells of the invention arealso a feature of the invention.

The invention also provides for knockout plants that comprise astaygreen potential phenotype. For example, the invention provides for aknockout plant that comprises a staygreen potential phenotype, where thestaygreen potential phenotype results from a disruption in at least oneendogenous ACC synthase gene. In one embodiment, the disruption includesone or more transposons, and inhibits expression or activity of at leastone ACC synthase protein compared to a corresponding control plant. Inanother embodiment, the disruption includes one or more point mutationsin the endogenous ACC synthase gene and inhibits expression or activityof at least one ACC synthase protein compared to a corresponding controlplant. In certain embodiments, the at least one endogenous ACC synthasegene comprises a nucleic acid sequence comprising, e.g., at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, about 99.5% ormore, sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), orSEQ ID NO:3 (gACS7), or a complement thereof. In certain embodiments,the knockout plant is a hybrid plant. Essentially all of the featuresnoted above apply to this embodiment as well, as relevant.

In another embodiment, a knockout plant includes a transgenic plant thatcomprises a staygreen potential phenotype. For example, a transgenicplant of the invention includes a staygreen potential phenotyperesulting from at least one introduced transgene that inhibits ethylenesynthesis. The introduced transgene includes a nucleic acid sequenceencoding at least one ACC synthase or subsequence thereof, which nucleicacid sequence comprises, e.g., at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, about 99.5% or more sequence identity toSEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ IDNO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7) or SEQ ID NO:10(CCRA178R), or a subsequence thereof, or a complement thereof, and is ina configuration that modifies a level of expression or activity of theat least one ACC synthase (e.g., a sense, antisense, RNA silencing orinterference configuration). Essentially all of the features noted aboveapply to this embodiment as well, as relevant.

A transgenic plant of the invention can also include a staygreenpotential phenotype resulting from at least one introduced transgenewhich inhibits ethylene synthesis, where said at least one introducedtransgene comprises a nucleic acid sequence encoding a subsequence(s) ofat least one ACC synthase, which at least one ACC synthase comprises,e.g., at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 99.5% or more sequence identity to SEQ ID NO:7 (pACS2), SEQID NO:8 (pACS6), SEQ ID NO.:9 (pACS7), or SEQ ID NO.:11 (pCCRA178R), ora conservative variation thereof. The nucleic acid sequence is typicallyin an RNA silencing or interference configuration (or, e.g., a sense orantisense configuration), and modifies a level of expression or activityof the at least one ACC synthase. Essentially all of the features notedabove apply to this embodiment as well, as relevant.

The staygreen potential of a plant of the invention includes, but is notlimited to, e.g., (a) a reduction in the production of at least one ACCsynthase specific mRNA; (b) a reduction in the production of an ACCsynthase; (c) a reduction in the production of ethylene; (d) a delay inleaf senescence; (e) an increase of drought resistance; (f) an increasedtime in maintaining photosynthetic activity; (g) an increasedtranspiration; (h) an increased stomatal conductance; (i) an increasedCO₂ assimilation; (j) an increased maintenance of CO₂ assimilation; or(k) any combination of (a)-(j); compared to a corresponding controlplant, and the like.

One aspect of the invention provides knockout or transgenic plantsincluding sterility phenotypes, e.g., a male or female sterilityphenotype. Thus, one class of embodiments provides a knockout plantcomprising a male sterility phenotype (e.g., reduced pollen shedding)which results from at least one disruption in at least one endogenousACC synthase gene. The disruption inhibits expression or activity of atleast one ACC synthase protein compared to a corresponding controlplant. In one embodiment, the at least one disruption results in reducedethylene production by the knockout plant as compared to the controlplant. In one embodiment, the at least one disruption includes one ormore transposons, wherein the one or more transposons are in the atleast one endogenous ACC synthase gene. In another embodiment, the atleast one disruption comprises one or more point mutations, wherein theone or more point mutations are in the at least one endogenous ACCsynthase gene. In yet another embodiment, the at least one disruption isintroduced into the knockout plant by introducing at least onepolynucleotide sequence comprising one or more subsequences of an ACCsynthase nucleic acid sequence configured for RNA silencing orinterference. In certain embodiments, the at least one endogenous ACCsynthase gene comprises a nucleic acid sequence comprising, e.g., atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about99.5% or more, sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2(gACS6), or SEQ ID NO:3 (gACS7), or a complement thereof. Essentiallyall of the features noted above apply to this embodiment as well, asrelevant.

Another class of embodiments provides a transgenic knockout plantcomprising a male sterility phenotype resulting from at least oneintroduced transgene which inhibits ethylene synthesis. The at least oneintroduced transgene comprises a nucleic acid sequence encoding at leastone ACC synthase, which nucleic acid sequence comprises at least about85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6(cACS7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or acomplement thereof, and is in a configuration that modifies a level ofexpression or activity of the at least one ACC synthase (e.g., anantisense, sense or RNA silencing or interference configuration). Incertain embodiments, the transgene includes a tissue-specific promoteror an inducible promoter. Essentially all of the features noted aboveapply to this embodiment as well, as relevant.

Polynucleotides are also a feature of the invention. In certainembodiments, an isolated or recombinant polynucleotide comprises amember selected from the group consisting of: (a) a polynucleotide, or acomplement thereof, comprising, e.g., at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 99.5% or more sequenceidentity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3(gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7)or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a conservativevariation thereof; (b) a polynucleotide, or a complement thereof,encoding a polypeptide sequence of SEQ ID NO:7 (pACS2), SEQ ID NO:8(pACS6), SEQ ID NO.:9 (pACS7), or SEQ ID NO:11 (pCCRA178R), or asubsequence thereof, or a conservative variation thereof; (c) apolynucleotide, or a complement thereof, that hybridizes under stringentconditions over substantially the entire length of a polynucleotidesubsequence comprising at least 100 contiguous nucleotides of SEQ IDNO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4(cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7), or SEQ ID NO:10(CCRA178R), or that hybridizes to a polynucleotide sequence of (a) or(b); and, (d) a polynucleotide that is at least about 85% identical to apolynucleotide sequence of (a), (b) or (c). In certain embodiments, thepolynucleotide inhibits ethylene production when expressed in a plant.

The polynucleotides of the invention can comprise or be contained withinan expression cassette or a vector (e.g., a viral vector). The vector orexpression cassette can comprise a promoter (e.g., a constitutive,tissue-specific, or inducible promoter) operably linked to thepolynucleotide. A polynucleotide of the invention can be linked to thepromoter in an antisense orientation or a sense orientation, beconfigured for RNA silencing or interference, or the like.

The invention also provides methods for inhibiting ethylene productionin a plant (and plants produced by such methods). For example, a methodof inhibiting ethylene production comprises inactivating one or more ACCsynthase genes in the plant, wherein the one or more ACC synthase genesencode one or more ACC synthases, wherein at least one of the one ormore ACC synthases comprises, e.g., at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 99.5% or more identity to SEQID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO:9 (pAC7) or SEQ ID NO:11(pCCRA178R).

In one embodiment, the inactivating step comprises introducing one ormore mutations into an ACC synthase gene sequence, wherein the one ormore mutations in the ACC synthase gene sequence comprise one or moretransposons, thereby inactivating the one or more ACC synthase genescompared to a corresponding control plant. In another embodiment, theinactivating step comprises introducing one or more mutations into anACC synthase gene sequence, wherein the one or more mutations in the ACCsynthase gene sequence comprise one or more point mutations, therebyinactivating the one or more ACC synthase genes compared to acorresponding control plant. The one or more mutations can comprise, forexample, a homozygous disruption in the one or more ACC synthase genes,a heterozygous disruption in the one or more ACC synthase genes, or acombination of both homozygous disruptions and heterozygous disruptionsif more than one ACC synthase gene is disrupted. In certain embodiments,the one or more mutations are introduced by a sexual cross. In certainembodiments, at least one of the one or more ACC synthase genes is,e.g., at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 99.5% or more, identical to SEQ ID NO:1 (gACS2), SEQ ID NO:2(gACS6) or SEQ ID NO:3 (gAC7), or a complement thereof).

In another embodiment, the inactivating step comprises: (a) introducinginto the plant at least one polynucleotide sequence, wherein the atleast one polynucleotide sequence comprises a nucleic acid encoding oneor more ACC synthases, or a subsequence thereof, and a promoter, whichpromoter functions in plants to produce an RNA sequence; and, (b)expressing the at least one polynucleotide sequence, therebyinactivating the one or more ACC synthase genes compared to acorresponding control plant (e.g., its non-transgenic parent or anon-transgenic plant of the same species). For example, the at least onepolynucleotide sequence can be introduced by techniques including, butnot limited to, electroporation, micro-projectile bombardment,Agrobacterium-mediated transfer, and the like. In certain aspects of theinvention, the polynucleotide is linked to the promoter in a senseorientation or an antisense orientation, or is configured for RNAsilencing or interference. Essentially all of the features noted aboveapply to this embodiment as well, as relevant.

Methods for modulating staygreen potential in plants are also a featureof the invention (as are plants produced by such methods). For example,a method of modulating staygreen potential comprises: a) selecting atleast one ACC synthase gene (e.g., encoding an ACC synthase, forexample, SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO:9 (pAC7) orSEQ ID NO:11 (pCCRA178R)) to mutate, thereby providing at least onedesired ACC synthase gene; b) introducing a mutant form (e.g., anantisense or sense configuration of at least one ACC synthase gene orsubsequence thereof, an RNA silencing configuration of at least one ACCsynthase gene or subsequence thereof, a heterozygous mutation in the atleast one ACC synthase gene, a homozygous mutation in the at least oneACC synthase gene or a combination of homozygous mutation andheterozygous mutation if more than one ACC synthase gene is selected,and the like) of the at least one desired ACC synthase gene into theplant; and, c) expressing the mutant form, thereby modulating staygreenpotential in the plant. In one embodiment, selecting the at least oneACC synthase gene comprises determining a degree (e.g., weak, moderateor strong) of staygreen potential desired. In certain embodiments, themutant gene is introduced by Agrobacterium-mediated transfer,electroporation, micro-projectile bombardment, a sexual cross, or thelike. Essentially all of the features noted above apply to thisembodiment as well, as relevant.

Detection of expression products is performed either qualitatively (bydetecting presence or absence of one or more product of interest) orquantitatively (by monitoring the level of expression of one or moreproduct of interest). In one embodiment, the expression product is anRNA expression product. Aspects of the invention optionally includemonitoring an expression level of a nucleic acid, polypeptide orchemical (e.g., ACC, ethylene, etc.) as noted herein for detection ofACC synthase, ethylene production, staygreen potential, etc. in a plantor in a population of plants.

The compositions and methods of the invention can include a variety ofplants, e.g., a plant of the Poaceae (Gramineae) family. Examples ofmembers of the Poaceae family include, by are not limited to,Acamptoclados, Achnatheruni, Achnella, Acroceras, Aegilops, Aegopgon,Agroelymus, Agrohordeum, Agropogon, Agropyron, Agrositanion, Agrostis,Aira, Allolepis, Alloteropsis, Alopecurus, Amblyopyrum, Ammophila,Ampelodesmos, Amphibromus, Amphicarpum, Amphilophis, Anastrophus,Anatherum, Andropogron, Anemathele, Aneurolepidium, Anisantha,Anthaenantia, Anthephora, Anthochloa, Anthoxanthum, Apera, Apluda,Archtagrostis, Arctophila, Argillochloa, Aristida, Arrhenatherum,Arthraxon, Arthrostylidium, Arundinaria, Arundinella, Arundo, Aspris,Atheropogon, Avena, Avenella, Avenochloa, Avenula, Axonopus, Bambusa,Beckmannia, Blepharidachne, Blepharoneuron, Bothriochloa, Bouteloua,Brachiaria, Brachyelytrum, Brachypodium, Briza, Brizopyrum, Bromelica,Bromopsis, Bromus, Buchloe, Bulbilis, Calamagrostis, Calamovilfa,Campulosus, Capriola, Catabrosa, Catapodium, Cathestecum, Cenchropsis,Cenchrus, Centotheca, Ceratochloa, Chaetochloa, Chasmanthium,Chimonobambusa, Chionochloa, Chloris, Chondrosum, Chrysopon, Chusquea,Cinna, Cladoraphis, Coelorachis, Coix, Coleanthus, Colpodium,Coridochloa, Cornucopiae, Cortaderia, Corynephorus, Cottea, Critesion,Crypsis, Ctenium, Cutandia, Cylindropyrum, Cymbopogon, Cynodon,Cynosurus, Cytrococcum, Dactylis, Dactyloctenium, Danthonia, Dasyochloa,Dasyprum, Davyella, Dendrocalamus, Deschampsia, Desmazeria, Deyeuxia,Diarina, Diarrhena, Dichanthelium, Dichanthium, Dichelachne, Diectomus,Digitaria, Dimeria, Dimorpostachys, Dinebra, Diplachne, Dissanthelium,Dissochondrus, Distichlis, Drepanostachyum, Dupoa, Dupontia,Echinochloa, Ectosperma, Ehrharta, Eleusine, Elyhordeum, Elyleymus,Elymordeum, Elymus, Elyonurus, Elysitanion, Elytesion, Elytrigia,Enneapogon, Enteropogon, Epicampes, Eragrostis, Eremochloa, Eremopoa,Eremopyrum, Erianthus, Ericoma, Erichloa, Eriochrysis, Erioneuron,Euchlaena, Euclasta, Eulalia, Eulaliopsis, Eustachys, Fargesia, Festuca,Festulolium, Fingerhuthia, Fluminia, Garnotia, Gastridium, Gaudinia,Gigantochloa, Glyceria, Graphephorum, Gymnopogon, Gynerium,Hackelochloa, Hainardia, Hakonechloa, Haynaldia, Heleochloa,Helictotrichon, Hemarthria, Hesperochloa, Hesperostipa, Heteropogon,Hibanobambusa, Hierochloe, Hilaria, Holcus, Homalocenchrus, Hordeum,Hydrochloa, Hymenachne, Hyparrhenia, Hypogynium, Hystrix, Ichnanthus,Imperata, Indocalamus, Isachne, Ischaemum, Ixophorus, Koeleria,Korycarpus, Lagurus, Lamarckia, Lasiacis, Leersia, Leptochloa,Leptochloopsis, Leptocoryphium, Leptoloma, Leptogon, Lepturus,Lerchenfeldia, Leucopoa, Leymostachys, Leymus, Limnodea, Lithachne,Lolium, Lophochlaena, Lophochloa, Lophopyrum, Ludolfia, Luziola,Lycurus, Lygeum, Maltea, Manisuris, Megastachya, Melica, Melinis,Mibora, Microchloa, Microlaena, Microstegium, Milium, Miscanihus,Mnesithea, Molinia, Monanthochloe, Monerma, Monroa, Muhlenbergia,Nardus, Nassella, Nazia, Neeragrostis, Neoschischkinia, Neostapfia,Neyraudia, Nothoholcus, Olyra, Opizia, Oplismenus, Orcuttia, Oryza,Oryzopsis, Otatea, Oxytenanthera, Panicularia, Panicum, Pappophorum,Parapholis, Pascopyrum, Paspalidium, Paspalum, Pennisetum, Phalaris,Phalaroides, Phanopyrum, Pharus, Phippsia, Phleum, Pholiurus,Phragmites, Phyllostachys, Piptatherum, Piptochaetium, Pleioblastus,Pleopogon, Pleuraphis, Pleuropogon, Poa, Podagrostis, Polypogon,Polytrias, Psathyrostachys, Pseudelymus, Pseudoroegneria, Pseudosasa,Ptilagrostis, Puccinellia, Pucciphippsia, Redfieldia, Reimaria,Reimarochloa, Rhaphis, Rhombolytrum, Rhynchelytrum, Roegneria,Rostraria, Rottboellia, Rytilix, Saccharum, Sacciolepis, Sasa, Sasaella,Sasamorpha, Savastana, Schedonnardus, Schismus, Schizachne,Schizachyrium, Schizostachyum, Sclerochloa, Scleropoa, Scleropogon,Scolochloa, Scribneria, Secale, Semiarundinaria, Sesleria, Setaria,Shibataea, Sieglingia, Sinarundinaria, Sinobambusa, Sinocalamus,Sitanion, Sorghastrum, Sorghum, Spartina, Sphenopholis, Spodiopogon,Sporobolus, Stapfia, Steinchisma, Stenotaphrum, Stipa, Stipagrostis,Stiporyzopsis, Swallenia, Syntherisma, Taeniatherum, Terrellia,Terrelymus, Thamnocalamus, Themeda, Thinopyrum, Thuarea, Thysanolaena,Torresia, Torreyochloa, Trachynia, Trachypogon, Tragus, Trichachne,Trichloris, Tricholaena, Trichoneura, Tridens, Triodia, Triplasis,Tripogon, Tripsacum, Trisetobromus, Trisetum, Triticosecale, Triticum,Tuctoria, Uniola, Urachne, Uralepis, Urochloa, Vahlodea, Valota,Vaseyochloa, Ventenata, Vetiveria, Vilfa, Vulpia, Willkommia, Yushania,Zea, Zizania, Zizaniopsis, and Zoysia. In one embodiment, the plant isZea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean,tomato, potato, pepper, broccoli, cabbage, a commercial corn line, orthe like.

Kits which incorporate one or more of the nucleic acids or polypeptidesnoted above are also a feature of the invention. Such kits can includeany of the above noted components and further include, e.g.,instructions for use of the components in any of the methods notedherein, packaging materials, containers for holding the components,and/or the like. For example, a kit for modulating staygreen potentialin a plant includes a container containing at least one polynucleotidesequence comprising a nucleic acid sequence, where the nucleic acidsequence is, e.g., at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 99.5% or more, identical to SEQ ID NO:1(gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2),SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cAC7) or SEQ ID NO:10 (CCRA178R), or asubsequence thereof, or a complement thereof. In a further embodiment,the kit includes instructional materials for the use of the at least onepolynucleotide sequence to control staygreen potential in a plant.Essentially all of the features noted above apply to this embodiment aswell, as relevant.

As another example, a kit for modulating sterility, e.g., malesterility, in a plant includes a container containing at least onepolynucleotide sequence comprising a nucleic acid sequence, wherein thenucleic acid sequence is, e.g., at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, about 99.5% or more, identical to SEQ IDNO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4(cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cAC7) or SEQ ID NO:10(CCRA178R), or a subsequence thereof, or a complement thereof. The kitoptionally also includes instructional materials for the use of the atleast one polynucleotide sequence to control sterility, e.g., malesterility, in a plant. Essentially all of the features noted above applyto this embodiment as well, as relevant.

DEFINITIONS

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular devices or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, the singular forms “a”, “an” and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a cell” includes acombination of two or more cells, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. In describing and claiming thepresent invention, the following terminology will be used in accordancewith the definitions set out below.

The term “plant” refers generically to any of: whole plants, plant partsor organs (e.g., leaves, stems, roots, etc.), shoot vegetativeorgans/structures (e.g. leaves, stems and tubers), roots, flowers andfloral organs/structures (e.g. bracts, sepals, petals, stamens, carpels,anthers and ovules), seed (including embryo, endosperm, and seed coat),fruit (the mature ovary), plant tissue (e.g. vascular tissue, groundtissue, and the like), tissue culture callus, and plant cells (e.g.guard cells, egg cells, trichomes and the like), and progeny of same.Plant cell, as used herein, further includes, without limitation, cellsobtained from or found in: seeds, cultures, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. Plant cells can alsobe understood to include modified cells, such as protoplasts, obtainedfrom the aforementioned tissues.

The term “dicot” refers to a dicotyledonous plant. Dicotyledonous plantsbelong to large subclass of Angiosperms that have two seed-leaves(cotyledon).

The term “monocot” refers to a monocotyledonous plant, which in thedeveloping plant has only one cotyledon.

The term “knockout plant cell” refers to a plant cell having adisruption in at least one ACC synthase gene in the cell, where thedisruption results in a reduced expression or activity of the ACCsynthase encoded by that gene compared to a control cell. The knockoutcan be the result of, e.g., antisense constructs, sense constructs, RNAsilencing constructs, RNA interference, genomic disruptions (e.g.,transposons, tilling, homologous recombination, etc.), and the like. Theterm “knockout plant” refers to a plant that has a disruption in atleast one of its ACC synthase genes in at least one cell.

The term “transgenic” refers to a plant that has incorporated nucleicacid sequences, including but not limited to genes, polynucleotides,DNA, RNA, etc., which have been introduced into a plant compared to anon-introduced plant.

The term “endogenous” relates to any gene or nucleic acid sequence thatis already present in a cell.

A “transposable element” (TE) or “transposable genetic element” is a DNAsequence that can move from one location to another in a cell. Movementof a transposable element can occur from episome to episome, fromepisome to chromosome, from chromosome to chromosome, or from chromosometo episome. Transposable elements are characterized by the presence ofinverted repeat sequences at their termini. Mobilization is mediatedenzymatically by a “transposase.” Structurally, a transposable elementis categorized as a “transposon,” (“TN”) or an “insertion sequenceelement,” (IS element) based on the presence or absence, respectively,of genetic sequences in addition to those necessary for mobilization ofthe element. A mini-transposon or mini-IS element typically lackssequences encoding a transposase.

The term “nucleic acid” or “polynucleotide” is generally used in itsart-recognized meaning to refer to a ribose nucleic acid (RNA) ordeoxyribose nucleic acid (DNA) polymer, or analog thereof, e.g., anucleotide polymer comprising modifications of the nucleotides, apeptide nucleic acid, or the like. In certain applications, the nucleicacid can be a polymer that includes multiple monomer types, e.g., bothRNA and DNA subunits. A nucleic acid can be, e.g., a chromosome orchromosomal segment, a vector (e.g., an expression vector), anexpression cassette, a naked DNA or RNA polymer, the product of apolymerase chain reaction (PCR), an oligonucleotide, a probe, etc. Anucleic acid can be e.g., single-stranded and/or double-stranded. Unlessotherwise indicated, a particular nucleic acid sequence of thisinvention optionally comprises or encodes complementary sequences, inaddition to any sequence explicitly indicated.

The term “polynucleotide sequence” or “nucleotide sequence” refers to acontiguous sequence of nucleotides in a single nucleic acid or to arepresentation, e.g., a character string, thereof. That is, a“polynucleotide sequence” is a polymer of nucleotides (anoligonucleotide, a DNA, a nucleic acid, etc.) or a character stringrepresenting a nucleotide polymer, depending on context. From anyspecified polynucleotide sequence, either the given nucleic acid or thecomplementary polynucleotide sequence (e.g., the complementary nucleicacid) can be determined.

The term “subsequence” or “fragment” is any portion of an entiresequence.

A “phenotype” is the display of a trait in an individual plant resultingfrom the interaction of gene expression and the environment.

An “expression cassette” is a nucleic acid construct, e.g., vector, suchas a plasmid, a viral vector, etc., capable of producing transcriptsand, potentially, polypeptides encoded by a polynucleotide sequence. Anexpression vector is capable of producing transcripts in an exogenouscell, e.g., a bacterial cell, or a plant cell, in vivo or in vitro,e.g., a cultured plant protoplast. Expression of a product can be eitherconstitutive or inducible depending, e.g., on the promoter selected.Antisense, sense or RNA interference or silencing configurations thatare not or cannot be translated are expressly included by thisdefinition. In the context of an expression vector, a promoter is saidto be “operably linked” to a polynucleotide sequence if it is capable ofregulating expression of the associated polynucleotide sequence. Theterm also applies to alternative exogenous gene constructs, such asexpressed or integrated transgenes. Similarly, the term operably linkedapplies equally to alternative or additional transcriptional regulatorysequences such as enhancers, associated with a polynucleotide sequence.

A polynucleotide sequence is said to “encode” a sense or antisense RNAmolecule, or RNA silencing or interference molecule or a polypeptide, ifthe polynucleotide sequence can be transcribed (in spliced or unsplicedform) and/or translated into the RNA or polypeptide, or a subsequencethereof.

“Expression of a gene” or “expression of a nucleic acid” meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing), translation of RNA into a polypeptide (possiblyincluding subsequent modification of the polypeptide, e.g.,posttranslational modification), or both transcription and translation,as indicated by the context.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Genes typically include coding sequencesand/or the regulatory sequences required for expression of such codingsequences. The term “gene” applies to a specific genomic sequence, aswell as to a cDNA or an mRNA encoded by that genomic sequence. Genesalso include non-expressed nucleic acid segments that, for example, formrecognition sequences for other proteins. Non-expressed regulatorysequences include promoters and enhancers, to which regulatory proteinssuch as transcription factors bind, resulting in transcription ofadjacent or nearby sequences.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can additionally comprisenon-amino acid elements such as labels, quenchers, blocking groups, orthe like and can optionally comprise modifications such as glycosylationor the like. The amino acid residues of the polypeptide can be naturalor non-natural and can be unsubstituted, unmodified, substituted ormodified.

The term “recombinant” indicates that the material (e.g., a cell, anucleic acid, or a protein) has been artificially or synthetically(non-naturally) altered by human intervention. The alteration can beperformed on the material within, or removed from, its naturalenvironment or state. For example, a “recombinant nucleic acid” is onethat is made by recombining nucleic acids, e.g., during cloning, DNAshuffling or other procedures; a “recombinant polypeptide” or“recombinant protein” is a polypeptide or protein which is produced byexpression of a recombinant nucleic acid. Examples of recombinant cellsinclude cells containing recombinant nucleic acids and/or recombinantpolypeptides.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophage,pro-viruses, phagemids, transposons, and artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that are not autonomously replicating.

In the context of the invention, the term “isolated” refers to abiological material, such as a nucleic acid or a protein, which issubstantially free from components that normally accompany or interactwith it in its naturally occurring environment. The isolated materialoptionally comprises material not found with the material in its naturalenvironment, e.g., a cell. For example, if the material is in itsnatural environment, such as a cell, the material has been placed at alocation in the cell (e.g., genome or genetic element) not native to amaterial found in that environment. For example, a naturally occurringnucleic acid (e.g., a coding sequence, a promoter, an enhancer, etc.)becomes isolated if it is introduced by non-naturally occurring means toa locus of the genome (e.g., a vector, such as a plasmid or virusvector, or amplicon) not native to that nucleic acid. An isolated plantcell, for example, can be in an environment (e.g., a cell culturesystem, or purified from cell culture) other than the native environmentof wild-type plant cells (e.g., a whole plant).

The term “variant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The variant can have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. Alternatively, a variantcan have “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Analogous minor variation can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software. Examplesof conservative substitutions are also described below.

A “host cell”, as used herein, is a cell which has been transformed ortransfected, or is capable of transformation or transfection, by anexogenous polynucleotide sequence. “Exogenous polynucleotide sequence”is defined to mean a sequence not naturally in the cell, or which isnaturally present in the cell but at a different genetic locus, indifferent copy number, or under direction of a different regulatoryelement.

A “promoter”, as used herein, includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells, such as Agrobacterium orRhizobium. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, or seeds or spatially in regions such asendosperm, embryo, or meristematic regions. Such promoters are referredto as “tissue-preferred” or “tissue-specific”. A temporally regulatedpromoter drives expression at particular times, such as between 0-25days after pollination. A “cell-type-preferred” promoter primarilydrives expression in certain cell types in one or more organs, forexample, vascular cells in roots or leaves. An “inducible” promoter is apromoter that is under environmental control and may be inducible orde-repressible. Examples of environmental conditions that may effecttranscription by inducible promoters include anaerobic conditions or thepresence of light. Tissue-specific, cell-type-specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions and in all or nearly all tissues, at all ornearly all stages of development.

“Transformation”, as used herein, is the process by which a cell is“transformed” by exogenous DNA when such exogenous DNA has beenintroduced inside the cell membrane. Exogenous DNA may or may not beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell. In prokaryotes and yeasts, for example, the exogenous DNAmay be maintained on an episomal element, such as a plasmid. Withrespect to higher eukaryotic cells, a stably transformed or transfectedcell is one in which the exogenous DNA has become integrated into thechromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates ethylene biosynthetic and signalinggenes in plants, e.g., Arabidopsis.

FIG. 2 schematically illustrates isolated and mapped ACC synthase genesand Mu insertion mutations. ACC6 is also known as ACS6, ACC2 is alsoknown as ACS2, and ACC7 is also known as ACS7.

FIG. 3, Panels A, B, C, and D illustrate heterozygous ACC synthaseknockouts in plants, e.g., maize. Panel A and Panel B illustrateheterozygous ACC synthase knockout plants in a field of wild-typeplants. Panel C and Panel D illustrate leaves from a heterozygous ACCsynthase knockout plant, left side of panel, compared to leaves from anACC synthase wild-type plant, right side of panel.

FIG. 4 illustrates an enhanced staygreen trait observed in leaves ofplants that are homozygous ACC synthase knockouts (right) compared towild type leaves (left) and heterozygous knockout leaves (middle).

FIG. 5, Panels A, B, C, D, E, and F illustrate leaf transpiration(Panels A and D), stomatal conductance (Panels B and E) and CO₂assimilation (Panels C and F) for wild-type (B73, +/+) and ACS6 null(15, O/O) mutant leaves under control conditions (Panels A, B, and C) ordrought conditions (Panels D, E, and F). For control conditions, plantswere grown under well-watered conditions and each leaf on a plant wasmeasured at forty days after pollination (dap). For drought conditions,plants were grown under limited water conditions and each leaf on aplant was measured at forty days after pollination. Values represent amean of six determinations.

FIG. 6, Panels A, B and C illustrate leaf transpiration (Panel A),stomatal conductance (Panel B) and CO₂ assimilation (Panel C) forwild-type (B73, +/+), ACS2 null (7, O/O) and ACS6 null (15, O/O) mutantleaves. Plants were grown under limited water conditions and each leafon a plant was measured at forty days after pollination. Valuesrepresent a mean of six determinations.

FIG. 7 schematically illustrates phylogenetic analysis of ACC synthasegene sequences, where maize sequences are indicated by (A47 (also knownas ACS2 or ACC2 herein), A50 (also known as ACS7 or ACC7 herein), A65(also known as ACS6 or ACC6 herein)), Arabidopsis sequences areindicated by (AtACS . . . ), tomato sequences are indicated by (LeACS .. . ), rice sequences are indicated by (indica (OsiACS . . . ), &japonica (OsjACS . . . )), wheat sequences are indicated by (TaACS . . .), and banana sequences are indicated by (MaACS . . . ).

FIG. 8 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), A50 (also knownas ACS7 or ACC7), and A65 (also known as ACS6 or ACC6) with both dicot(AtACS, LeACS) and monocot (OsiACS, OsjACS, TaACS, and MaACS) species.The alignment is done with a most stringent criteria (identical aminoacids) and the plurality is 26.00, the threshold is 4, the AveWeight is1.00, the AveMatch is 2.78 and the AvMisMatch is −2.25. SEQ ID NOs areas follows: A47pep SEQ ID NO:25; A50pep SEQ ID NO:26; osiacs1pep SEQ IDNO:27; osjacs1pep SEQ ID NO:28; TaACS2pep SEQ ID NO:29; AtACS1pep SEQ IDNO:30; AtACS2pep SEQ ID NO:31; LeACS2pep SEQ ID NO:32; LeACS4pep SEQ IDNO:33; MaACS1pep SEQ ID NO:34; MaACS5pep SEQ ID NO:35; LeACS1Apep SEQ IDNO:36; LeACS1Bpep SEQ ID NO:37; LeACS6pep SEQ ID NO:38; AtACS6pep SEQ IDNO:39; A65pep SEQ ID NO:40; osiacs2pep SEQ ID NO:41; AtACS5pep SEQ IDNO:42; AtACS9 SEQ ID NO:43; AtACS4pep SEQ ID NO:44; AtACS8 SEQ ID NO:45;MaACS2pep SEQ ID NO:46; MaACS3pep SEQ ID NO:47; LeACS3pep SEQ ID NO:48;LeACS7pep SEQ ID NO:49; OsjACS2pep SEQ ID NO:50; OsjACS3 SEQ ID NO:51;OsiACS3pep SEQ ID NO:52; and AtACS7 SEQ ID NO:53.

FIG. 9 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), A50 (also knownas ACS7 or ACC7), and A65 (also known as ACS6 or ACC6) with both dicot(AtACS, LeACS) and monocot (OsiACS, OsjACS, TaACS, MaACS) species (SEQID NOs:25-53). The alignment is done with a stringent criteria (similaramino acid residues) and the plurality is 26.00, the threshold is 2, theAveWeight is 1.00, the AveMatch is 2.78 and the AvMisMatch is −2.25.

FIG. 10 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), A50 (also knownas ACS7 or ACC7), and A65 (also known as ACS6 or ACC6) with both dicot(AtACS, LeACS) and monocot (OsiACS, OsjACS, TaACS, MaACS) species (SEQID NOs:25-53). The alignment is done with a less stringent criteria(somewhat similar amino acid residues) and the plurality is 26.00, thethreshold is 0, the AveWeight is 1.00, the AveMatch is 2.78 and theAvMisMatch is −2.25.

FIG. 11 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), and A50 (alsoknown as ACS7 or ACC7) with sequences that are most similar to ACS2 andACS7 (SEQ ID NOs:25-39). The alignment is done with most stringentcriteria (identical amino acids) and the plurality is 15.00, thethreshold is 4, the AveWeight is 1.00, the AveMatch is 2.78 and theAvMisMatch is −2.25.

FIG. 12 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), and A50 (alsoknown as ACS7 or ACC7) with sequences that are most similar to ACS2 andACS7 (SEQ ID NOs:25-39). The alignment is done with stringent criteria(similar amino acid residues) and the plurality is 15.00, the thresholdis 2, the AveWeight is 1.00, the AveMatch is 2.78 and the AvMisMatch is−2.25.

FIG. 13 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A65 (also known as ACS6 or ACC6) with sequencesthat are most similar to ACS6 (SEQ ID NOs:40-53). The alignment is donewith most stringent criteria (identical amino acids) and the pluralityis 14.00, the threshold is 4, the AveWeight is 1.00, the AveMatch is2.78 and the AvMisMatch is −2.25.

FIG. 14 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A65 (also known as ACS6 or ACC6) with sequencesthat are most similar to ACS6 (SEQ ID NOs:40-53). The alignment is donewith stringent criteria (similar amino acid residues) and the pluralityis 14.00, the threshold is 2, the AveWeight is 1.00, the AveMatch is2.78 and the AvMisMatch is −2.25.

FIG. 15 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), A50 (also knownas ACS7 or ACC7), and A65 (also known as ACS6 or ACC6) (SEQ ID NOs:25-26and 40). The alignment is done with most stringent criteria (identicalamino acids) and the plurality is 3.00, the threshold is 4, theAveWeight is 1.00, the AveMatch is 2.78 and the AvMisMatch is −2.25.

FIG. 16 illustrates a peptide consensus sequence alignment with ACCsynthase sequences of A47 (also known as ACS2 or ACC2), A50 (also knownas ACS7 or ACC7), and A65 (also known as ACS6 or ACC6) (SEQ ID NOs:25-26and 40). The alignment is done with stringent criteria (similar aminoacid residues) and the plurality is 3.00, the threshold is 2, theAveWeight is 1.00, the AveMatch is 2.78 and the AvMisMatch is −2.25.

FIG. 17 Panels A, B, C, and D illustrate total chlorophyll data forwild-type and ACC synthase knockout plants. Panels A and B illustratetotal chlorophyll data for wild-type (B73, +/+), ACS2 null (0/0), andACS6 null (0/0) plants 40 days after pollination for plants grown undernormal conditions (Panel A) or drought conditions (Panel B). Panel Ccompares total chlorophyll for wild-type (B73, +/+) and ACS6 null (0/0)plants 40 days after pollination under normal and drought conditions.Panel D illustrates a comparison of total chlorophyll for B73(wild-type) plants collected at 30 and 40 days after pollination.

FIG. 18 Panels A, B, C, and D illustrate soluble protein data forwild-type and ACC synthase knockout plants. Panels A and B illustratesoluble protein data for wild-type (B73, +/+), ACS2 null (0/0), and ACS6null (0/0) plants 40 days after pollination for plants grown undernormal conditions (Panel A) or drought conditions (Panel B). Panel Ccompares soluble protein for wild-type (B73, +/+) and ACS6 null (0/0)plants 40 days after pollination under normal and drought conditions.Panel D illustrates a comparison of soluble protein for B73 (wild-type)plants collected at 30 and 40 days after pollination.

FIG. 19, Panels A and B illustrate ethylene production in seedlingleaves. Panel A illustrates various lines. In Panel B, the seedlingleaves are averaged by genotype. In Panel C, ethylene production wasdetermined for every leaf of wild-type (i.e., B73) plants at 20, 30, and40 DAP. Leaf 1 represents the oldest surviving leaf and leaf 11 theyoungest. Three replicates were measured and the average and standarddeviation reported.

FIG. 20 Panel A illustrates chlorophyll data, Panel B soluble protein,and Panel C Rubisco expression. The level of chlorophyll a+b (Panel A)and soluble protein (Panel B) was measured in the third oldest leaf(Leaf 3), sixth oldest leaf (Leaf 6), and ninth oldest leaf (Leaf 9) ofadult wild-type (i.e., ACS6/ACS6), acs2/acs2, and acs6/acs6 plantsfollowing dark treatment for 7 days. Plants were watered daily.Additional acs6/acs6 plants were watered daily with 100 μM ACC duringthe treatment. acs6/acs6 leaves watered with 100 μM ACC but keptunsheathed are also shown. The average and standard deviation of leavesfrom three individual plants is shown. (Panel C) Western analysis of thesame leaves was performed using rice anti-Rubisco antiserum. Solubleprotein from leaf samples of equal fresh weight was used.

FIG. 21 Panels A-C illustrate the ACS2 hairpin construct. Panel A is aschematic diagram of PHP20600 containing a ubiquitin promoter (UBI1ZMPRO) driving expression of the ACS2 hairpin (a terminal repeatconsisting of TR1 and TR2). RB represents the Agrobacterium right bordersequence. A 4126 bp fragment of the 49682 bp cassette is illustrated.Panel B presents the sequence of ZM-ACS2 TR1 (SEQ ID NO:54), and Panel Cpresents the sequence of ZM-ACS2 TR2 (SEQ ID NO:55).

FIG. 22 Panels A-C illustrate the ACS6 hairpin construct. Panel A is aschematic diagram of PHP20323 containing a ubiquitin promoter (UBI1ZMPRO) driving expression of the ACS6 hairpin (a terminal repeatconsisting of TR1 and TR2). RB represents the Agrobacterium right bordersequence. A 3564 bp fragment of the 49108 bp cassette is illustrated.Panel B presents the sequence of ZM-ACS6 TR1 (SEQ ID NO:56), and Panel Cpresents the sequence of ZM-ACS6 TR2 (SEQ ID NO:57).

FIG. 23 Panels A and B illustrate events generated for ACS2- andACS6-hairpin constructs. Panel A presents a diagram showing the numberof individual events for ACS2 hairpin (PHP20600) and the associatedtransgene copy number per event. Panel B presents a diagram showing thenumber of individual events for ACS6 hairpin (PHP20323) and theassociated transgene copy number per event.

DETAILED DESCRIPTION

“Stay-green” is a term commonly used to describe a plant phenotype.Staygreen is a desirable trait in commercial agriculture, e.g., adesirable trait associated with grain filling. As described herein, fivefundamentally distinct types of stay-green have been described,including Types A, B, C, D, and E (see, e.g., Thomas H and Smart C M(1993) Crops that stay green. Annals of Applied Biology 123:193-219; andThomas H and Howarth C J (2000) Five ways to stay green. Journal ofExperimental Botany 51:329-337). However, there is very littledescription of the biochemical, physiological or molecular basis forgenetically determined stay-green. See, e.g., Thomas and Howarth, supra.This invention provides a molecular/biochemical basis for staygreenpotential.

A number of environmental and physiological conditions have been shownto significantly alter the timing and progression of leaf senescence andcan provide some insight into the basis for this trait. Amongenvironmental factors, light is probably the most significant, and ithas long been established that leaf senescence can be induced in manyplant species by placing detached leaves in darkness. See, e.g., WeaverL M and Amasino R M (2001) Senescence is induced in individuallydarkened Arabidopsis leaves, but inhibited in whole darkened plants.Plant Physiology 127:876-886. Limited nutrient and water availabilityhave also been shown to induce leaf senescence prematurely. See, e.g.,Rosenow D T, et al. (1983) Drought-tolerant sorghum and cottongermplasm. Agricultural Water Management 7:207-222. Among physiologicaldeterminants, growth regulators play a key role in directing the leafsenescence program. Modification of cytokinin levels can significantlydelay leaf senescence. For example, plants transformed with isopentenyltransferase (ipt), an Agrobacterium gene encoding a rate-limiting stepin cytokinin biosynthesis, when placed under the control of a senescenceinducible promoter, resulted in autoregulated cytokinin production and astrong stay-green phenotype. See, e.g., Gan S and Amasino R M (1995)Inhibition of leaf senescence by autoregulated production of cytokinin.Science 270:1986-1988. However, there are other factors that areinvolved with this trait.

For example, ethylene has also been implicated in controlling leafsenescence (see, e.g., Davis K M and Grierson D (1989) Identification ofcDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs thataccumulate during fruit ripening and leaf senescence in response toethylene. Planta 179:73-80) and some dicot plants impaired in ethyleneproduction or perception also show a delay in leaf senescence (see,e.g., Picton S, et al. (1993) Altered fruit ripening and leaf senescencein tomatoes expressing an antisense ethylene-forming enzyme transgene.The Plant Journal 3:469-481; Grbic V and Bleeker A B (1995) Ethyleneregulates the timing of leaf senescence in Arabidopsis. The PlantJournal 8:95-102; and, John I, et al. (1995) Delayed leaf senescence inethylene-deficient ACC-oxidase antisense tomato plants: molecular andphysiological analysis. The Plant Journal 7:483-490), which can bephenocopied by exogenous application of inhibitors of ethylenebiosynthesis and action (see, e.g., Abeles F B, et al. (1992) Ethylenein Plant Biology. Academic Press, San Diego, Calif.).

Ethylene perception involves membrane-localized receptors that, e.g., inArabidopsis include ETR1, ERS1, ETR2, ERS2, and EIN4 (see FIG. 1). ETR1,ETR2, and EIN4 are composed of three domains, an N-terminal ethylenebinding domain, a putative histidine protein kinase domain, and aC-terminal receiver domain, whereas ERS1 and ERS2 lack the receiverdomain. These genes have been grouped into two subfamilies based onhomology, where ETR1 and ERS1 comprise one subfamily and ETR2, ERS2 andEIN4 comprise the other. In Arabidopsis, analysis of loss-of-functionmutants has revealed that ethylene inhibits the signaling activity ofthese receptors and subsequently their ability to activate CTR1, anegative regulator of ethylene responses that is related to mammalianRAF-type serine/threonine kinases. Ethylene signal transduction pathwaysuggests that ethylene binding to the receptor inhibits its own kinaseactivity, resulting in decreased activity of CTR1, and consequently, anincrease in EIN2 activity (which acts downstream of CTR1) ultimatelyleads to increases in ethylene responsiveness. Differential expressionof members of the ethylene receptor family has been observed, bothdevelopmentally and in response to ethylene.

The identification and analysis of mutants in Arabidopsis and tomatothat are deficient in ethylene biosynthesis and perception are valuablein establishing the role that ethylene plays in plant growth anddevelopment. Mutant analysis has also been instrumental in identifyingand characterizing the ethylene signal transduction pathway. While manyethylene mutants have been identified in dicot plants (e.g., Arabidopsisand tomato), no such mutants have been identified in monocots (e.g.,rice, wheat, and corn). Herein are described, e.g., ethylene mutants(e.g., in a monocot) deficient in ACC synthase, the first enzyme in theethylene biosynthetic pathway.

This invention provides ACC synthase polynucleotide sequences fromplants, which modulate staygreen potential in plants and ethyleneproduction, exemplified by, e.g., SEQ ID NO:1 through SEQ ID NO:6 andSEQ ID NO:10 and, e.g., a set of polypeptide sequences which modulatestaygreen potential in plants and/or ethylene production, e.g., SEQ IDNO:7 through SEQ ID NO:9 and SEQ ID NO:11. The invention also providesknockout plant cells deficient in ACC synthase and knockout plantshaving a staygreen potential phenotype, as well as knockout plantshaving a male sterility phenotype. The plants of the invention can havethe characteristic of regulating responses to environmental stressbetter than control plants, e.g., higher tolerance to drought stress.Plants of the invention can also have a higher tolerance for otherstresses (e.g., crowding in, e.g., maize) compared to a control plants.Thus, plants of the invention can be planted at higher densities thancurrently practiced by farmers. In addition, plants of the invention arecritical in elucidating the regulatory roles that ethylene playsthroughout plant development as well as its role in regulating responsesto stress, e.g., drought, crowding, etc.

Ethylene in Plants

Ethylene (C₂H₄) is a gaseous plant hormone. It has a varied spectrum ofeffects that can be tissue and/or species specific. For example,physiological activities include, but are not limited to, promotion offood ripening, abscission of leaves and fruit of dicotyledonous species,flower senescence, stem extension of aquatic plants, gas space(aerenchyma) development in roots, leaf epinastic curvatures, stem andshoot swelling (in association with stunting), femaleness in curcubits,fruit growth in certain species, apical hook closure in etiolatedshoots, root hair formation, flowering in the Bromeliaceae,diageotropism of etiolated shoots, and increased gene expression (e.g.,of polygalacturonase, cellulase, chitinases, β-1,3-glucanases, etc.).Ethylene is released naturally by ripening fruit and is also produced bymost plant tissues, e.g., in response to stress (e.g., drought,crowding, disease or pathogen attack, temperature (cold or heat) stress,wounding, etc.), and in maturing and senescing organs.

Ethylene is generated from methionine by a well-defined pathwayinvolving the conversion of S-adenosyl-L-methionine (SAM or Ado Met) tothe cyclic amino acid 1-aminocyclopropane-1-carboxylic acid (ACC) whichis facilitated by ACC synthase (see, e.g., FIG. 1). Sulphur is conservedin the process by recycling 5′-methythioadenosine.

ACC synthase is an aminotransferase which catalyzes the rate limitingstep in the formation of ethylene by converting S-adenosylmethionine toACC. Typically, the enzyme requires pyridoxal phosphate as a cofactor.ACC synthase is typically encoded in multigene families. Examplesinclude SEQ ID NOs:1-3, described herein. Individual members can exhibittissue-specific regulation and/or are induced in response toenvironmental and chemical stimuli. Features of the invention includeACC synthase sequences and subsequences. See the section herein entitled“Polynucleotides and Polypeptides of the Invention.”

Ethylene is then produced from the oxidation of ACC through the actionof ACC oxidase (also known as the ethylene forming enzyme) with hydrogencyanide as a secondary product that is detoxified by β-cyanoalaninesynthase. ACC oxidase is encoded by multigene families in whichindividual members exhibit tissue-specific regulation and/or are inducedin response to environmental and chemical stimuli. Activity of ACCoxidase can be inhibited by anoxia and cobalt ions. The ACC oxidaseenzyme is stereospecific and uses cofactors, e.g., Fe⁺², O₂, ascorbate,etc. Finally, ethylene is metabolized by oxidation to CO₂ or to ethyleneoxide and ethylene glycol.

Polynucleotides and Polypeptides of the Invention

The invention features the identification of gene sequences, codingnucleic acid sequences, and amino acid sequences of ACC synthase, whichare associated, e.g., with staygreen potential in plants and/or ethyleneproduction. The sequences of the invention can influence staygreenpotential in plants by modulating the production of ethylene.

Polynucleotide sequences of the invention include, e.g., thepolynucleotide sequences represented by SEQ ID NO:1 through SEQ ID NO:6and SEQ ID NO:10, and subsequences thereof. In addition to the sequencesexpressly provided in the accompanying sequence listing, the inventionincludes polynucleotide sequences that are highly related structurallyand/or functionally. For example, polynucleotides encoding polypeptidesequences represented by SEQ ID NO:7 through SEQ ID NO:9 and SEQ IDNO:11, or subsequences thereof, are one embodiment of the invention. Inaddition, polynucleotide sequences of the invention includepolynucleotide sequences that hybridize under stringent conditions to apolynucleotide sequence comprising any of SEQ ID NO:1-SEQ ID NO:6 andSEQ ID NO:10, or a subsequence thereof (e.g., a subsequence comprisingat least 100 contiguous nucleotides). Polynucleotides of the inventionalso include ACC synthase sequences and/or subsequences configured forRNA production, e.g., mRNA, antisense RNA, sense RNA, RNA silencing andinterference configurations, etc.

In addition to the polynucleotide sequences of the invention, e.g.,enumerated in SEQ ID NO:1 to SEQ ID NO:6 and SEQ ID NO:10,polynucleotide sequences that are substantially identical to apolynucleotide of the invention can be used in the compositions andmethods of the invention. Substantially identical or substantiallysimilar polynucleotide sequences are defined as polynucleotide sequencesthat are identical, on a nucleotide by nucleotide bases, with at least asubsequence of a reference polynucleotide, e.g., selected from SEQ IDNOs:1-6 and 10. Such polynucleotides can include, e.g., insertions,deletions, and substitutions relative to any of SEQ ID NOs:1-6 and 10.For example, such polynucleotides are typically at least about 70%identical to a reference polynucleotide selected from among SEQ ID NO:1through SEQ ID NO:6 and SEQ ID NO:10, or subsequence thereof. Forexample, at least 7 out of 10 nucleotides within a window of comparisonare identical to the reference sequence selected, e.g., from SEQ IDNO:1-6 and 10. Frequently, such sequences are at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 98%, at least about 99%, or atleast about 99.5%, identical to the reference sequence, e.g., at leastone of SEQ ID NO:1 to SEQ ID NO:6 or SEQ ID NO:10. Subsequences of thepolynucleotides of the invention described above, e.g., SEQ ID NOs:1-6and 10, including, e.g., at least about 5, at least about 10, at leastabout 15, at least about 20, at least about 25, at least about 50, atleast about 75, at least about 100, at least about 500, about 1000 ormore, contiguous nucleotides or complementary subsequences thereof arealso a feature of the invention. Such subsequences can be, e.g.,oligonucleotides, such as synthetic oligonucleotides, isolatedoligonucleotides, or full-length genes or cDNAs.

In addition, polynucleotide sequences complementary to any of the abovedescribed sequences are included among the polynucleotides of theinvention.

Polypeptide sequences of the invention include, e.g., the amino acidsequences represented by SEQ ID NO:7 through SEQ ID NO:9 and SEQ IDNO:11, and subsequences thereof. In addition to the sequences expresslyprovided in the accompanying sequence listing, the invention includesamino acid sequences that are highly related structurally and/orfunctionally. For example, in addition to the amino acid sequences ofthe invention, e.g., enumerated in SEQ ID NO:7 to SEQ ID NO:9 and SEQ IDNO:11, amino acid sequences that are substantially identical to apolypeptide of the invention can be used in the compositions and methodsof the invention. Substantially identical or substantially similar aminoacid sequences are defined as amino acid sequences that are identical,on an amino acid by amino acid bases, with at least a subsequence of areference polypeptide, e.g., selected from SEQ ID NOs:7-9 and 11. Suchpolypeptides can include, e.g., insertions, deletions, and substitutionsrelative to any of SEQ ID NOs:7-9 and 11. For example, such polypeptidesare typically at least about 70% identical to a reference polypeptideselected from among SEQ ID NO:7 through SEQ ID NO:9 and SEQ ID NO:11, ora subsequence thereof. For example, at least 7 out of 10 amino acidswithin a window of comparison are identical to the reference sequenceselected, e.g., from SEQ ID NO:7-9 and 11. Frequently, such sequencesare at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,at least about 99%, or at least about 99.5%, identical to the referencesequence, e.g., at least one of SEQ ID NO:7 to SEQ ID NO:9 or SEQ IDNO:11. Subsequences of the polypeptides of the invention describedabove, e.g., SEQ ID NOs:7-9 and 11, including, e.g., at least about 5,at least about 10, at least about 15, at least about 20, at least about25, at least about 50, at least about 75, at least about 100, at leastabout 500, about 1000 or more, contiguous amino acids are also a featureof the invention. Conservative variants of amino acid sequences orsubsequences of the invention are also amino acid sequences of theinvention. Polypeptides of the invention are optionally immunogenic,enzymatically active, enzymatically inactive, and the like.

Where the polynucleotide sequences of the invention are translated toform a polypeptide or subsequence of a polypeptide, nucleotide changescan result in either conservative or non-conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having functionally similar side chains.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Table 1 sets forth six groups whichcontain amino acids that are “conservative substitutions” for oneanother. Other conservative substitution charts are available in theart, and can be used in a similar manner. TABLE 1 ConservativeSubstitution Group 1 Alanine (A) Serine (S) Threonine (T) 2 Asparticacid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

One of skill in the art will appreciate that many conservativesubstitutions of the nucleic acid constructs which are disclosed yieldfunctionally identical constructs. For example, as discussed above,owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10% or more) are substituted with different amino acids with highlysimilar properties, are also readily identified as being highly similarto a disclosed construct. Such conservative variations of each disclosedsequence are a feature of the invention.

Methods for obtaining conservative variants, as well as more divergentversions of the nucleic acids and polypeptides of the invention, arewidely known in the art. In addition to naturally occurring homologueswhich can be obtained, e.g., by screening genomic or expressionlibraries according to any of a variety of well-established protocols,see, e.g., Ausubel et al. Current Protocols in Molecular Biology(supplemented through 2004) John Wiley & Sons, New York (“Ausubel”);Sambrook et al. Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”), and Berger and Kimmel Guide to Molecular CloningTechniques, Methods in Enzymology volume 152 Academic Press, Inc., SanDiego, Calif. (“Berger”), additional variants can be produced by any ofa variety of mutagenesis procedures. Many such procedures are known inthe art, including site directed mutagenesis, oligonucleotide-directedmutagenesis, and many others. For example, site directed mutagenesis isdescribed, e.g., in Smith (1985) “In vitro mutagenesis” Ann. Rev. Genet.19:423-462, and references therein, Botstein & Shortle (1985)“Strategies and applications of in vitro mutagenesis” Science229:1193-1201; and Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7. Oligonucleotide-directed mutagenesis is described, e.g., inZoller & Smith (1982) “Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment” Nucleic Acids Res.10:6487-6500). Mutagenesis using modified bases is described e.g., inKunkel (1985) “Rapid and efficient site-specific mutagenesis withoutphenotypic selection” Proc. Natl. Acad. Sci. USA 82:488-492, and Tayloret al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13:8765-8787. Mutagenesis using gapped duplex DNA isdescribed, e.g., in Kramer et al. (1984) “The gapped duplex DNA approachto oligonucleotide-directed mutation construction” Nucl. Acids Res.12:9441-9460). Point mismatch mutagenesis is described, e.g., by Krameret al. (1984) “Point Mismatch Repair” Cell 38:879-887). Double-strandbreak mutagenesis is described, e.g., in Mandecki (1986)“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177-7181, and in Arnold (1993) “Protein engineeringfor unusual environments” Current Opinion in Biotechnology 4:450-455).Mutagenesis using repair-deficient host strains is described, e.g., inCarter et al. (1985) “Improved oligonucleotide site-directed mutagenesisusing M13 vectors” Nucl. Acids Res. 13:4431-4443. Mutagenesis by totalgene synthesis is described e.g., by Nambiar et al. (1984) “Totalsynthesis and cloning of a gene coding for the ribonuclease S protein”Science 223:1299-1301. DNA shuffling is described, e.g., by Stemmer(1994) “Rapid evolution of a protein in vitro by DNA shuffling” Nature370:389-391, and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution.” Proc.Natl. Acad. Sci. USA 91:10747-10751.

Many of the above methods are further described in Methods in EnzymologyVolume 154, which also describes useful controls for trouble-shootingproblems with various mutagenesis methods. Kits for mutagenesis, libraryconstruction and other diversity generation methods are alsocommercially available. For example, kits are available from, e.g.,Amersham International plc (Piscataway, N.J.) (e.g., using the Ecksteinmethod above), Bio/Can Scientific (Mississauga, Ontario, CANADA),Bio-Rad (Hercules, Calif.) (e.g., using the Kunkel method describedabove), Boehringer Mannheim Corp. (Ridgefield, Conn.), ClonetechLaboratories of BD Biosciences (Palo Alto, Calif.), DNA Technologies(Gaithersburg, Md.), Epicentre Technologies (Madison, Wis.) (e.g., the 5prime 3 prime kit); Genpak Inc. (Stony Brook, N.Y.), Lemargo Inc(Toronto, CANADA), Invitrogen Life Technologies (Carlsbad, Calif.), NewEngland Biolabs (Beverly, Mass.), Pharmacia Biotech (Peapack, N.J.),Promega Corp. (Madison, Wis.), QBiogene (Carlsbad, Calif.), andStratagene (La Jolla, Calif.) (e.g., QuickChange™ site-directedmutagenesis kit and Chameleon™ double-stranded, site-directedmutagenesis kit).

Determining Sequence Relationships

The nucleic acid and amino acid sequences of the invention include,e.g., those provided in SEQ ID NO:1 to SEQ ID NO:11 and subsequencesthereof, as well as similar sequences. Similar sequences are objectivelydetermined by any number of methods, e.g., percent identity,hybridization, immunologically, and the like. A variety of methods fordetermining relationships between two or more sequences (e.g., identity,similarity and/or homology) are available and well known in the art. Themethods include manual alignment, computer assisted sequence alignmentand combinations thereof, for example. A number of algorithms (which aregenerally computer implemented) for performing sequence alignment arewidely available or can be produced by one of skill. These methodsinclude, e.g., the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2:482; the homology alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443; the search for similarity methodof Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444;and/or by computerized implementations of these algorithms (e.g., GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

For example, software for performing sequence identity (and sequencesimilarity) analysis using the BLAST algorithm is described in Altschulet al. (1990) J. Mol. Biol. 215:403-410. This software is publiclyavailable, e.g., through the National Center for BiotechnologyInformation on the world wide web at ncbi.nlm.nih.gov. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold. These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP (BLAST Protein)program uses as defaults a wordlength (W) of 3, an expectation (E) of10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

Additionally, the BLAST algorithm performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul (1993)Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (p(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence(and, therefore, in this context, homologous) if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, or less than about 0.01, and oreven less than about 0.001.

Another example of a useful sequence alignment algorithm is PILEUP.PILEUP creates a multiple sequence alignment from a group of relatedsequences using progressive, pairwise alignments. It can also plot atree showing the clustering relationships used to create the alignment.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similarto the method described by Higgins & Sharp (1989) CABIOS5:151-153. Theprogram can align, e.g., up to 300 sequences of a maximum length of5,000 letters. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster can then be aligned to the next mostrelated sequence or cluster of aligned sequences. Two clusters ofsequences can be aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program can also be used toplot a dendogram or tree representation of clustering relationships. Theprogram is run by designating specific sequences and their amino acid ornucleotide coordinates for regions of sequence comparison.

An additional example of an algorithm that is suitable for multiple DNA,or amino acid, sequence alignments is the CLUSTALW program (Thompson, J.D. et al. (1994) Nucl. Acids. Res. 22:4673-4680). CLUSTALW performsmultiple pairwise comparisons between groups of sequences and assemblesthem into a multiple alignment based on homology. Gap open and Gapextension penalties can be, e.g., 10 and 0.05 respectively. For aminoacid alignments, the BLOSUM algorithm can be used as a protein weightmatrix. See, e.g., Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.USA 89: 10915-10919.

Nucleic Acid Hybridization

Similarity between ACC synthase nucleic acids of the invention can alsobe evaluated by “hybridization” between single stranded (or singlestranded regions of) nucleic acids with complementary or partiallycomplementary polynucleotide sequences.

Hybridization is a measure of the physical association between nucleicacids, typically, in solution, or with one of the nucleic acid strandsimmobilized on a solid support, e.g., a membrane, a bead, a chip, afilter, etc. Nucleic acid hybridization occurs based on a variety ofwell characterized physico-chemical forces, such as hydrogen bonding,solvent exclusion, base stacking, and the like. Numerous protocols fornucleic acid hybridization are well known in the art. An extensive guideto the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” (Elsevier, N.Y.), as well as in Ausubel et al.Current Protocols in Molecular Biology (supplemented through 2004) JohnWiley & Sons, New York (“Ausubel”); Sambrook et al. Molecular Cloning—ALaboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (“Sambrook”), and Berger and Kimmel Guideto Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif. (“Berger”). Hames and Higgins(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2, IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

Conditions suitable for obtaining hybridization, including differentialhybridization, are selected according to the theoretical meltingtemperature (T_(m)) between complementary and partially complementarynucleic acids. Under a given set of conditions, e.g., solventcomposition, ionic strength, etc., the T_(m) is the temperature at whichthe duplex between the hybridizing nucleic acid strands is 50%denatured. That is, the T_(m) corresponds to the temperaturecorresponding to the midpoint in transition from helix to random coil;it depends on the length of the polynucleotides, nucleotide composition,and ionic strength, for long stretches of nucleotides.

After hybridization, unhybridized nucleic acids can be removed by aseries of washes, the stringency of which can be adjusted depending uponthe desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the T_(m)) lower the background signal, typically withprimarily the specific signal remaining. See, also, Rapley, R. andWalker, J. M. eds., Molecular Biomethods Handbook (Humana Press, Inc.1998).

“Stringent hybridization wash conditions” or “stringent conditions” inthe context of nucleic acid hybridization experiments, such as Southernand northern hybridizations, are sequence dependent, and are differentunder different environmental parameters. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993), supra, and inHames and Higgins 1 and Hames and Higgins 2, supra.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 2×SSC, 50%formamide at 42° C., with the hybridization being carried out overnight(e.g., for approximately 20 hours). An example of stringent washconditions is a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook,supra for a description of SSC buffer). Often, the wash determining thestringency is preceded by a low stringency wash to remove signal due toresidual unhybridized probe. An example low stringency wash is 2×SSC atroom temperature (e.g., 20° C. for 15 minutes).

In general, a signal to noise ratio of at least 2.5×-5× (and typicallyhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Detection of at least stringent hybridization between two sequences inthe context of the invention indicates relatively strong structuralsimilarity to, e.g., the nucleic acids of ACC synthases provided in thesequence listings herein.

For purposes of the invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. or lesslower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH (as noted below, highly stringentconditions can also be referred to in comparative terms). Targetsequences that are closely related or identical to the nucleotidesequence of interest (e.g., “probe”) can be identified under stringentor highly stringent conditions. Lower stringency conditions areappropriate for sequences that are less complementary.

For example, in determining stringent or highly stringent hybridization(or even more stringent hybridization) and wash conditions, thestringency of the hybridization and wash conditions is graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration, and/or increasing theconcentration of organic solvents, such as formamide, in thehybridization or wash), until a selected set of criteria are met. Forexample, the stringency of the hybridization and wash conditions isgradually increased until a probe comprising one or more polynucleotidesequences of the invention, e.g., selected from SEQ ID NO:1 to SEQ IDNO:6 and SEQ ID NO:10, or a subsequence thereof, and/or complementarypolynucleotide sequences thereof, binds to a perfectly matchedcomplementary target (again, a nucleic acid comprising one or morenucleic acid sequences or subsequences selected from SEQ ID NO:1 to SEQID NO:6 and SEQ ID NO:10, and complementary polynucleotide sequencesthereof), with a signal to noise ratio that is at least 2.5×, andoptionally 5×, or 10×, or 100× or more, as high as that observed forhybridization of the probe to an unmatched target, as desired.

Using subsequences derived from the nucleic acids encoding the ACCsynthase polypeptides of the invention, novel target nucleic acids canbe obtained; such target nucleic acids are also a feature of theinvention. For example, such target nucleic acids include sequences thathybridize under stringent conditions to an oligonucleotide probe thatcorresponds to a unique subsequence of any of the polynucleotides of theinvention, e.g., SEQ ID NOs:1-6, 10, or a complementary sequencethereof; the probe optionally encodes a unique subsequence in any of thepolypeptides of the invention, e.g., SEQ ID NOs:7-9 and 11.

For example, hybridization conditions are chosen under which a targetoligonucleotide that is perfectly complementary to the oligonucleotideprobe hybridizes to the probe with at least about a 5-10× higher signalto noise ratio than for hybridization of the target oligonucleotide to anegative control non-complimentary nucleic acid.

Higher ratios of signal to noise can be achieved by increasing thestringency of the hybridization conditions such that ratios of about15×, 20×, 30×, 50× or more are obtained. The particular signal willdepend on the label used in the relevant assay, e.g., a fluorescentlabel, a calorimetric label, a radioactive label, or the like.

Vectors, Promoters and Expression Systems

Nucleic acids of the invention can be in any of a variety of forms,e.g., expression cassettes, vectors, plasmids, or linear nucleic acidsequences. For example, vectors, plasmids, cosmids, bacterial artificialchromosomes (BACs), YACs (yeast artificial chromosomes), phage, virusesand nucleic acid segments can comprise an ACC synthase nucleic acidsequence or subsequence thereof which one desires to introduce intocells. These nucleic acid constructs can further include promoters,enhancers, polylinkers, regulatory genes, etc.

Thus, the present invention also relates, e.g., to vectors comprisingthe polynucleotides of the present invention, host cells thatincorporate the vectors of the invention, and the production ofpolypeptides of the invention by recombinant techniques.

In accordance with this aspect of the invention, the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, ora single or double-stranded RNA or DNA viral vector. Such vectors may beintroduced into cells as polynucleotides, preferably DNA, by well knowntechniques for introducing DNA and RNA into cells. The vectors, in thecase of phage and viral vectors, also may be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host, operably linked to the polynucleotide to beexpressed. Appropriate trans-acting factors are supplied by the host,supplied by a complementing vector or supplied by the vector itself uponintroduction into the host.

In certain preferred embodiments in this regard, the vectors provide forpreferred expression. Such preferred expression may be inducibleexpression, temporally limited expression, or expression restricted topredominantly certain types of cells, or any combination of the above.Particularly preferred among inducible vectors are vectors that can beinduced for expression by environmental factors that are easy tomanipulate, such as temperature and nutrient additives. A variety ofvectors suitable to this aspect of the invention, including constitutiveand inducible expression vectors for use in prokaryotic and eukaryotichosts, are well known and employed routinely by those of skill in theart. Such vectors include, among others, chromosomal, episomal andvirus-derived vectors, e.g., vectors derived from bacterial plasmids,from bacteriophage, from transposons, from yeast episomes, frominsertion elements, from yeast chromosomal elements, from viruses suchas baculoviruses, papova viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids and binaries used for Agrobacterium-mediated transformations.All may be used for expression in accordance with this aspect of thepresent invention.

Vectors that are functional in plants can be binary plasmids derivedfrom Agrobacterium. Such vectors are capable of transforming plantcells. These vectors contain left and right border sequences that arerequired for integration into the host (plant) chromosome. At minimum,between these border sequences is the gene (or other polynucleotidesequence of the present invention) to be expressed, typically undercontrol of regulatory elements. In one embodiment, a selectable markerand a reporter gene are also included. For ease of obtaining sufficientquantities of vector, a bacterial origin that allows replication in E.coli can be used.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Useful plant binary vectorsinclude BIN19 and its derivatives available from Clontech. These vectorsare listed solely by way of illustration of the many commerciallyavailable and well-known vectors that are available to those of skill inthe art for use in accordance with this aspect of the present invention.It will be appreciated that any other plasmid or vector suitable for,for example, introduction, maintenance, propagation or expression of apolynucleotide or polypeptide of the invention in a host may be used inthis aspect of the invention, several of which are disclosed in moredetail below.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, aribosome-binding site for translation when the construct encodes apolypeptide. The coding portion of the mature transcripts expressed bythe constructs will include a translation-initiating AUG at thebeginning and a termination codon appropriately positioned at the end ofthe polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination signals, among others. For secretion of a translatedprotein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Transcription of the DNA (e.g., encoding the polypeptides) of thepresent invention by higher eukaryotes may be increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp that act to increasetranscriptional activity of a promoter in a given host cell-type.Examples of enhancers include the SV40 enhancer, which is located on thelate side of the replication origin at bp 100 to 270, thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.Additional enhancers useful in the invention to increase transcriptionof the introduced DNA segment, include, inter alia, viral enhancers likethose within the 35S promoter, as shown by Odell et al., Plant Mol.Biol. 10:263-72 (1988), and an enhancer from an opine gene as describedby Fromm et al., Plant Cell 1:977 (1989). The enhancer may affect thetissue-specificity and/or temporal specificity of expression ofsequences included in the vector.

Termination regions also facilitate effective expression by endingtranscription at appropriate points. Useful terminators for practicingthis invention include, but are not limited to, pinII (see An et al.,Plant Cell 1(1):115-122 (1989)), glb1 (see Genbank Accession #L22345),gz (see gzw64a terminator, Genbank Accession #S78780), and the nosterminator from Agrobacterium. The termination region can be native withthe promoter nucleotide sequence, can be native with the DNA sequence ofinterest, or can be derived from another source. For example, otherconvenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also: Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Among known eukaryotic promoters suitable for generalized expression arethe CMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), metallothionein promoters, suchas the mouse metallothionein-I promoter and various plant promoters,such as globulin-1. When available, the native promoters of the ACCsynthase genes may be used. Representatives of prokaryotic promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters to name just a few of the well-known promoters.

Isolated or recombinant plants, or plant cells, incorporating the ACCsynthase nucleic acids are a feature of the invention. Thetransformation of plant cells and protoplasts can be carried out inessentially any of the various ways known to those skilled in the art ofplant molecular biology, including, but not limited to, the methodsdescribed herein. See, in general, Methods in Enzymology, Vol. 153(Recombinant DNA Part D) Wu and Grossman (eds.) 1987, Academic Press,incorporated herein by reference. As used herein, the term“transformation” means alteration of the genotype of a host plant by theintroduction of a nucleic acid sequence, e.g., a “heterologous”,“exogenous” or “foreign” nucleic acid sequence. The heterologous nucleicacid sequence need not necessarily originate from a different source butit will, at some point, have been external to the cell into which isintroduced.

In addition to Berger, Ausubel and Sambrook, useful general referencesfor plant cell cloning, culture and regeneration include Jones (ed)(1995) Plant Gene Transfer and Expression Protocols—Methods in MolecularBiology, Volume 49 Humana Press Towata N.J.; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y. (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) (Gamborg). A variety ofcell culture media are described in Atlas and Parks (eds) The Handbookof Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas).Additional information for plant cell culture is found in availablecommercial literature such as the Life Science Research Cell CultureCatalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo) (Sigma-LSRCCC)and, e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-PCCS). Additional detailsregarding plant cell culture are found in Croy, (ed.) (1993) PlantMolecular Biology Bios Scientific Publishers, Oxford, U.K. See also thesection herein entitled “Plant transformations.”

In an embodiment of this invention, recombinant vectors including one ormore of the ACC synthase nucleic acids or a subsequence thereof, e.g.,selected from SEQ ID NO:1 to SEQ ID NO:6, or SEQ ID NO:10, suitable forthe transformation of plant cells are prepared. In another embodiment, anucleic acid sequence encoding for the desired ACC synthase RNA orprotein or subsequence thereof, e.g., selected from among SEQ ID NO:7 toSEQ ID NO:9, or SEQ ID NO:11, is conveniently used to construct arecombinant expression cassette which can be introduced into the desiredplant. In the context of the invention, an expression cassette willtypically comprise a selected ACC synthase nucleic acid sequence orsubsequence in an RNA configuration (e.g., antisense, sense, RNAsilencing or interference configuration, and/or the like) operablylinked to a promoter sequence and other transcriptional andtranslational initiation regulatory sequences which are sufficient todirect the transcription of the ACC synthase RNA configuration sequencein the intended tissues (e.g., entire plant, leaves, anthers, roots,etc.) of the transformed plant.

In general, the particular promoter used in the expression cassette inplants depends on the intended application. Any of a number of promoterscan be suitable. For example, the nucleic acids can be combined withconstitutive, inducible, tissue-specific (tissue-preferred), or otherpromoters for expression in plants. For example, a strongly or weaklyconstitutive plant promoter that directs expression of an ACC synthaseRNA configuration sequence in all tissues of a plant can be favorablyemployed. Such promoters are active under most environmental conditionsand states of development or cell differentiation. Examples ofconstitutive promoters include the 1′- or 2′-promoter of Agrobacteriumtumefaciens (see, e.g., O'Grady (1995) Plant Mol. Biol. 29:99-108).Other plant promoters include the ribulose-1,3-bisphosphate carboxylasesmall subunit promoter, the phaseolin promoter, alcohol dehydrogenase(Adh) gene promoters (see, e.g., Millar (1996) Plant Mol. Biol.31:897-904), sucrose synthase promoters, α-tubulin promoters, actinpromoters, such as the Arabidopsis actin gene promoter (see, e.g., Huang(1997) Plant Mol. Biol. 1997 33:125-139), cab, PEPCase, R gene complex,ACT11 from Arabidopsis (Huang et al. Plant Mol. Biol. 33:125-139(1996)), Cat3 from Arabidopsis (Zhong et al., Mol. Gen. Genet.251:196-203 (1996)), the gene encoding stearoyl-acyl carrier proteindesaturase from Brassica napus (Solocombe et al. (1994) Plant Physiol.104:1167-1176), GPc1 from maize (Martinez et al. (1989) J. Mol. Biol208:551-565), Gpc2 from maize (Manjunath et al. (1997), Plant Mol. Biol.33:97-112), and other transcription initiation regions from variousplant genes known to those of skill. See also Holtorf (1995) “Comparisonof different constitutive and inducible promoters for the overexpressionof transgenes in Arabidopsis thaliana,” Plant Mol. Biol. 29:637-646. Thepromoter sequence from the E8 gene (see, Deikman and Fischer (1988) EMBOJ 7:3315) and other genes can also be used, along with promotersspecific for monocotyledonous species (e.g., McElroy D., et al. (1994.)Foreign gene expression in transgenic cereals. Trends Biotech.,12:62-68). Other constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter and other constitutive promotersdisclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35Spromoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroyet al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al.(1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) PlantMol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet.81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALSpromoter (U.S. Pat. No. 5,659,026), and the like. Yet, otherconstitutive promoters include, for example, U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142; and 6,177,611.

In addition to the promoters noted herein, promoters of bacterial originwhich operate in plants can be used in the invention. They include,e.g., the octopine synthase promoter, the nopaline synthase promoter andother promoters derived from Ti plasmids. See, Herrera-Estrella et al.(1983) Nature 303:209. Viral promoters can also be used. Examples ofviral promoters include the 35S and 19S RNA promoters of cauliflowermosaic virus (CaMV). See, Odell et al., (1985) Nature 313:810; and,Dagless (1997) Arch. Virol. 142:183-191. Other examples of constitutivepromoters from viruses which infect plants include the promoter of thetobacco mosaic virus; cauliflower mosaic virus (CaMV) 19S and 35Spromoters or the promoter of Figwort mosaic virus, e.g., the figwortmosaic virus 35S promoter (see, e.g., Maiti (1997) Transgenic Res.6:143-156), etc. Alternatively, novel promoters with usefulcharacteristics can be identified from any viral, bacterial, or plantsource by methods, including sequence analysis, enhancer or promotertrapping, and the like, known in the art.

Tissue-preferred (tissue-specific) promoters and enhancers can beutilized to target enhanced gene expression within a particular planttissue. Tissue-preferred (tissue-specific) promoters include, e.g.,those described in Yamamoto et al. (1997) Plant J. 12(2):255-265;Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al.(1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) TransgenicRes. 6(2):157-168; Rinehart et al. (1996) Plant Physiol.112(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535;Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. CellDiffer. 20:181-196; Orozco et al. (1993) Plant Mol Biol.23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

In certain embodiments, leaf specific promoters can be used, e.g.,pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize),cab-m1 Ca+2 promoter from maize, the Arabidopsis thaliana myb-relatedgene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS)promoters (e.g., the tomato RBCS1, RBCS2 and RBCS3A genes, which areexpressed in leaves and light-grown seedlings, while RBCS1 and RBCS2 areexpressed in developing tomato fruits, and/or a ribulose bisphosphatecarboxylase promoter which is expressed almost exclusively in mesophyllcells in leaf blades and leaf sheaths at high levels, etc.), and thelike. See, e.g., Matsuoka et a., (1993) Tissue-specific light-regulatedexpression directed by the promoter of a C4 gene, maize pyruvate,orthophosphate dikinase, in a C3 plant, rice, PNAS USA 90(20):9586-90;(2000) Plant Cell Physiol. 41(1):42-48; (2001) Plant Mol. Biol.45(1):1-15; Shiina, T. et al., (1997) Identification of PromoterElements involved in the cytosolic Ca+2 mediated photoregulation ofmaize cab-m1 expression, Plant Physiol. 115:477-483; Casal (1998) PlantPhysiol. 116:1533-1538; Li (1996) FEBS Lett. 379:117-121; Busk (1997)Plant J. 11:1285-1295; and, Meier (1997) FEBS Lett. 415:91-95; and,Matsuoka (1994) Plant J. 6:311-319. Other leaf-specific promotersinclude, for example, Yamamoto et al. (1997) Plant J. 12(2):255-265;Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994)Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J.3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; andMatsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

In certain embodiments, senescence specific promoters can be used (e.g.,a tomato promoter active during fruit ripening, senescence andabscission of leaves, a maize promoter of gene encoding a cysteineprotease, and the like). See, e.g., Blume (1997) Plant J. 12:731-746;Griffiths et al., (1997) Sequencing, expression pattern and RFLP mappingof a senescence-enhanced cDNA from Zea Mays with high homology tooryzain gamma and aleurain, Plant Mol. Biol. 34(5):815-21; Zea mayspartial see1 gene for cysteine protease, promoter region and 5′ codingregion, Genbank AJ494982; Kleber-Janke, T. and Krupinska, K. (1997)Isolation of cDNA clones for genes showing enhanced expression in barleyleaves during dark-induced senescence as well as during senescence underfield conditions, Planta 203(3): 332-40; and, Lee, R H et al., (2001)Leaf senescence in rice plants: cloning and characterization ofsenescence up-regulated genes, J. Exp. Bot. 52(358):1117-21.

In other embodiments, anther-specific promoters can be used. Suchpromoters are known in the art or can be discovered by known techniques;see, e.g., Bhalla and Singh (1999) Molecular control of male fertilityin Brassica Proc. 10^(th) Annual Rapeseed Congress, Canberra, Australia;van Tunen et al. (1990) Pollen- and anther-specific chi promoters frompetunia: tandem promoter regulation of the chiA gene. Plant Cell2:393-40; Jeon et al. (1999) Isolation and characterization of ananther-specific gene, RA8, from rice (Oryza sativa L). Plant MolecularBiology 39:35-44; and Twell et al. (1993) Activation and developmentalregulation of an Arabidopsis anther-specific promoter in microspores andpollen of Nicotiana tabacum. Sex. Plant Reprod. 6:217-224.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue (see, e.g., EMBO J. 8(2):343-350).The TR1′ gene, fused to nptII (neomycin phosphotransferase II) showedsimilar characteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol.29(4):759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol.25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363;5,459,252; 5,401,836; 5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, e.g., Thompson et al.(1989) BioEssays 10:108, herein incorporated by reference. Suchseed-preferred promoters include, but are not limited to, Cim1(cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps(myo-inositol-1-phosphate synthase); mZE40-2, also known as Zm-40 (U.S.Pat. No. 6,403,862); nuc1c (U.S. Pat. No. 6,407,315); and celA(cellulose synthase) (see WO 00/11177). Gama-zein is anendosperm-specific promoter. Glob-1 is an embryo-specific promoter. Fordicots, seed-specific promoters include, but are not limited to, beanβ-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, a maize 15 kDa zein promoter, a 22 kDa zein promoter, a 27 kDa zeinpromoter, a g-zein promoter, a 27 kD γ-zein promoter (such as gzw64Apromoter, see Genbank Accession #S78780), a waxy promoter, a shrunken 1promoter, a shrunken 2 promoter, a globulin 1 promoter (see GenbankAccession #L22344), an ltp2 promoter (Kalla, et al., Plant Journal6:849-860 (1994); U.S. Pat. No. 5,525,716), cim1 promoter (see U.S. Pat.No. 6,225,529) maize end1 and end2 promoters (See U.S. Pat. No.6,528,704 and application Ser. No. 10/310,191, filed Dec. 4, 2002); nuc1promoter (U.S. Pat. No 6,407,315); Zm40 promoter (U.S. Pat. No.6,403,862); eep1 and eep2; lec1 (U.S. patent application Ser. No.09/718,754); thioredoxinH promoter (U.S. provisional patent applicationNo. 60/514,123); mlip15 promoter (U.S. Pat. No. 6,479,734); PCNA2promoter; and the shrunken-2 promoter. (Shaw et al., Plant Phys98:1214-1216, 1992; Zhong Chen et al., PNAS USA 100:3525-3530, 2003)However, other promoters useful in the practice of the invention areknown to those of skill in the art such as nucellain promoter ( See C.Linnestad, et al., Nucellain, A Barley Homolog of the DicotVacuolar—Processing Proteasem Is Localized in Nucellar Cell Walls, PlantPhysiol. 118:1169-80 (1998), kn1 promoter (See S. Hake and N. Ori, TheRole of knotted1 in Meristem Functions, B8: INTERACTIONS ANDINTERSECTIONS IN PLANT PATHWAYS, COEUR D'ALENE, IDAHO, KEYSTONESYMPOSIA, Feb. 8-14, 1999, at 27.), and F3.7 promoter (Baszczynski etal., Maydica 42:189-201 (1997)), etc. In certain embodiments, spatiallyacting promoters such as glb1, an embryo-preferred promoter; or gammazein, an endosperm-preferred promoter; or a promoter active in theembryo-surrounding region (see U.S. patent application Ser. No.10/786,679, filed Feb. 25, 2004), or BETL1 (See G. Hueros, et al., PlantPhysiology 121:1143-1152 (1999) and Plant Cell 7:747-57 (June 1995)),are useful, including promoters preferentially active in femalereproductive tissues, and those active in meristematic tissues,particularly in meristematic female reproductive tissues. See also, WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed.

A tissue-specific promoter can drive expression of operably linkedsequences in tissues other than the target tissue. Thus, as used herein,a tissue-specific promoter is one that drives expression preferentiallyin the target tissue, but can also lead to some expression in othertissues as well.

The use of temporally-acting promoters is also contemplated by thisinvention. For example, promoters that act from 0-25 days afterpollination (DAP), 4-21, 4-12, or 8-12 DAP can be selected, e.g.,promoters such as cim1 and ltp2. Promoters that act from −14 to 0 daysafter pollination can also be used, such as SAG12 (See WO 96/29858,Richard M. Amasino, published 3 Oct. 1996) and ZAG1 or ZAG2 (See R. J.Schmidt, et al., Identification and Molecular Characterization of ZAG1,the Maize Homolog of the Arabidopsis Floral Homeotic Gene AGAMOUS,Plant-Cell 5(7): 729-37 (July 1993)). Other useful promoters includemaize zag2.1, Zap (also known as ZmMADS; U.S. patent application Ser.No. 10/387,937; WO 03/078590); and the maize tb1 promoter (see alsoHubbarda et al., Genetics 162:1927-1935, 2002).

Where overexpression of an ACC synthase RNA configuration nucleic acidis detrimental to the plant, one of skill will recognize that weakconstitutive promoters can be used for low-levels of expression (or, incertain embodiments, inducible or tissue-specific promoters can beused). In those cases where high levels of expression are not harmful tothe plant, a strong promoter, e.g., a t-RNA, or other pol III promoter,or a strong pol II promoter (e.g., the cauliflower mosaic viruspromoter, CaMV, 35S promoter), can be used.

Where low level expression is desired, weak promoters will be used.Generally, by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By low level is intendedat levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts. Alternatively, it is recognized that weakpromoters also encompass promoters that drive expression in only a fewcells and not in others to give a total low level of expression. Where apromoter drives expression at unacceptably high levels, portions of thepromoter sequence can be deleted or modified to decrease expressionlevels. Such weak constitutive promoters include, for example, the corepromoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No.6,072,050), the core 35S CaMV promoter, and the like.

In certain embodiments of the invention, an inducible promoter can beused. For example, a plant promoter can be under environmental control.Such promoters are referred to as “inducible” promoters. Examples ofenvironmental conditions that can alter transcription by induciblepromoters include pathogen attack, anaerobic conditions, elevatedtemperature, and the presence of light. For example, the inventionincorporates the drought-inducible promoter of maize (Busk (1997) PlantJ. 11:1285-1295); the cold, drought, high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897-909), and the like.

Pathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi et al. (1983) Neth. J. PlantPathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and VanLoon (1985) Plant Mol. Virol. 4:111-116. See also the applicationentitled “Inducible Maize Promoters”, U.S. patent application Ser. No.09/257,583, filed Feb. 25, 1999.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; and Yang(1996) Proc. Natl. Acad. Sci. 93:14972-14977. See also, Chen et al.(1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad. Sci.91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertz et al.(1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the polynucleotidesof the invention. For example, the invention can use the auxin-responseelements E1 promoter subsequence (AuxREs) from the soybean (Glycine maxL.) (Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10:955-966); theauxin-inducible parC promoter from tobacco; a plant biotin responseelement (Streit (1997) Mol. Plant Microbe Interact. 10:933-937); and thepromoter responsive to the stress hormone abscisic acid (Sheen (1996)Science 274:1900-1902).

Plant promoters which are inducible upon exposure to chemical reagentswhich can be applied to the plant, such as herbicides or antibiotics,are also used to express the polynucleotides of the invention. Dependingupon the objective, the promoter can be a chemical-inducible promoter,where application of the chemical induces gene expression, or achemical-repressible promoter, where application of the chemicalrepresses gene expression. For example, the maize In2-2 promoter,activated by benzenesulfonamide herbicide safeners, can be used (DeVeylder (1997) Plant Cell Physiol. 38:568-577); application of differentherbicide safeners induces distinct gene expression patterns, includingexpression in the root, hydathodes, and the shoot apical meristem. AnACC synthase coding sequence or RNA configuration can also be under thecontrol of, e.g., tetracycline-inducible and tetracycline-repressiblepromoters (see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237;U.S. Pat. Nos. 5,814,618 and 5,789,156; and, Masgrau (1997) Plant J.11:465-473 (describing transgenic tobacco plants containing the Avenasativa L. (oat) arginine decarboxylase gene with atetracycline-inducible promoter); or, a salicylic acid-responsiveelement (Stange (1997) Plant J. 11:1315-1324. Other chemical-induciblepromoters are known in the art and include, but are not limited to, themaize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257).

Endogenous promoters of genes related to herbicide tolerance and relatedphenotypes are also useful for driving expression of ACC synthase RNAconfiguration nucleic acids, e.g., P450 monooxygenases,glutathione-S-transferases, homoglutathione-S-transferases, glyphosateoxidases and 5-enolpyruvylshikimate-2-phosphate synthases. For example,a plant promoter attached to a polynucleotide of the invention can beuseful when one wants to turn on expression in the presence of aparticular condition, e.g., drought conditions, short growingconditions, density, etc.

Tissue specific promoters can also be used to direct expression ofpolynucleotides of the invention, including the ACC synthase RNAconfiguration nucleic acids, such that a polynucleotide of the inventionis expressed only in certain tissues or stages of development, e.g.,leaves, anthers, roots, shoots, etc. Tissue specific expression can beadvantageous, for example, when expression of the polynucleotide incertain tissues is desirable while expression in other tissues isundesirable. Tissue specific promoters are transcriptional controlelements that are only active in particular cells or tissues at specifictimes during plant development, such as in vegetative tissues orreproductive tissues. Examples of tissue-specific promoters underdevelopmental control include promoters that initiate transcription only(or primarily only) in certain tissues, such as vegetative tissues,e.g., roots or leaves, or reproductive tissues, such as fruit, ovules,seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductivetissue-specific promoters may be, e.g., anther-specific, ovule-specific,embryo-specific, endosperm-specific, integument-specific, seed and seedcoat-specific, pollen-specific, petal-specific, sepal-specific, or somecombination thereof.

It will be understood that numerous promoters not mentioned are suitablefor use in this aspect of the invention, are well known and readily maybe employed by those of skill in the manner illustrated by thediscussion and the examples herein. For example, this inventioncontemplates using, when appropriate, the native ACC synthase promotersto drive the expression of the enzyme (or of ACC synthase polynucleotidesequences or subsequences) in a recombinant environment.

In preparing expression vectors of the invention, sequences other thanthose associated with the endogenous ACC synthase gene, mRNA orpolypeptide sequence, or subsequence thereof, can optionally be used.For example, other regulatory elements such as introns, leadersequences, polyadenylation regions, signal/localization peptides, etc.can also be included.

The vector comprising a polynucleotide of the invention can also includea marker gene which confers a selectable phenotype on plant cells. Forexample, the marker can encode biocide tolerance, particularlyantibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin,hygromycin, or herbicide tolerance, such as tolerance to chlorosulfuron,or phophinothricin. Reporter genes which are used to monitor geneexpression and protein localization via visualizable reaction products(e.g., beta-glucuronidase, beta-galactosidase, and chloramphenicolacetyltransferase) or by direct visualization of the gene product itself(e.g., green fluorescent protein, GFP; Sheen et al. (1995) The PlantJournal 8:777) can be used for, e.g., monitoring transient geneexpression in plant cells.

Vectors for propagation and expression generally will include selectablemarkers. Such markers also may be suitable for amplification or thevectors may contain additional markers for this purpose. In this regard,the expression vectors preferably contain one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells. Preferred markers include dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, and tetracycline or ampicillinresistance genes for culturing E. coli and other prokaryotes. Kanamycinand herbicide resistance genes (PAT and BAR) are generally useful inplant systems.

Selectable marker genes, in physical proximity to the introduced DNAsegment, are used to allow transformed cells to be recovered by eitherpositive genetic selection or screening. The selectable marker genesalso allow for maintaining selection pressure on a transgenic plantpopulation, to ensure that the introduced DNA segment, and itscontrolling promoters and enhancers, are retained by the transgenicplant.

Many of the commonly used positive selectable marker genes for planttransformation have been isolated from bacteria and code for enzymesthat metabolically detoxify a selective chemical agent which may be anantibiotic or a herbicide. Other positive selection marker genes encodean altered target which is insensitive to the inhibitor.

An example of a selection marker gene for plant transformation is theBAR or PAT gene, which is used with the selecting agent bialaphos(Spencer et al., J. Theor. Appl'd Genetics 79:625-631 (1990)). Anotheruseful selection marker gene is the neomycin phosphotransferase II(nptII) gene, isolated from Tn5, which confers resistance to kanamycinwhen placed under the control of plant regulatory signals (Fraley etal., Proc. Nat'l Acad. Sci. (USA) 80:4803 (1983)). The hygromycinphosphotransferase gene, which confers resistance to the antibiotichygromycin, is a further example of a useful selectable marker (VandenElzen et al., Plant Mol. Biol. 5:299 (1985)). Additional positiveselectable marker genes of bacterial origin that confer resistance toantibiotics include gentamicin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyl transferase and thebleomycin resistance determinant (Hayford et al., Plant Physiol. 86:1216(1988); Jones et al., Mol. Gen. Genet. 210:86 (1987); Svab et al., PlantMol. Biol. 14:197 (1990); Hille et al., Plant Mol. Biol. 7:171 (1986)).

Other positive selectable marker genes for plant transformation are notof bacterial origin. These genes include mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987); Shahet al., Science 233:478 (1986); Charest et al., Plant Cell Rep. 8:643(1990)). Other examples of suitable selectable marker genes include, butare not limited to: genes encoding resistance to chloramphenicol,Herrera Estrella et al. (1983) EMBO J. 2:987-992; methotrexate, HerreraEstrella et al. (1983) Nature 303:209-213; Meijer et al. (1991) PlantMol. Biol. 16:807-820; hygromycin, Waldron et al. (1985) Plant Mol.Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227;streptomycin, Jones et al. (1987) Mol. Gen. Genet. 210:86-91;spectinomycin, Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137;bleomycin, Hille et al. (1990) Plant Mol. Biol. 7:171-176; sulfonamide,Guerineau et al. (1990) Plant Mol. Biol. 15:127-136; bromoxynil, Stalkeret al. (1988) Science 242:419-423; glyphosate, Shaw et al. (1986)Science 233:478-481; phosphinothricin, DeBlock et al. (1987) EMBO J.6:2513-2518.

Another class of useful marker genes for plant transformation with theDNA sequence requires screening of presumptively transformed plant cellsrather than direct genetic selection of transformed cells for resistanceto a toxic substance such as an antibiotic. These genes are particularlyuseful to quantitate or visualize the spatial pattern of expression ofthe DNA sequence in specific tissues and are frequently referred to asreporter genes because they can be fused to a gene or gene regulatorysequence for the investigation of gene expression. Commonly used genesfor screening presumptively transformed cells include β-glucuronidase(GUS), β-galactosidase, luciferase, and chloramphenicolacetyltransferase (Jefferson, Plant Mol. Biol. Rep. 5:387 (1987); Teeriet al., EMBO J. 8:343 (1989); Koncz et al., Proc. Nat'l Acad. Sci. (USA)84:131 (1987); De Block et al., EMBO J. 3:1681 (1984)). Examples ofother suitable reporter genes known in the art can be found in, forexample: Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al.(1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J.9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu etal. (1996) Current Biology 6:325-330. Another approach to theidentification of relatively rare transformation events has been use ofa gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway (Ludwig et al., Science 247:449(1990)).

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. The sequence may be inserted in a forward or reverseorientation. Procedures for restriction and ligation that can be used tothis end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseof skill, are set forth in great detail in Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

A polynucleotide of the invention, optionally encoding the heterologousstructural sequence of a polypeptide of the invention, generally will beinserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. (Operably linked, asused herein, includes reference to a functional linkage between apromoter and a second sequence, wherein the promoter sequence initiatesand mediates transcription of the DNA corresponding to the secondsequence. Generally, operably linked means that the nucleic acidsequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.) Whenthe polynucleotide is intended for expression of a polypeptide, thepolynucleotide will be positioned so that the transcription start siteis located appropriately 5′ to a ribosome binding site. Theribosome-binding site will be 5′ to the AUG that initiates translationof the polypeptide to be expressed. Generally, there will be no otheropen reading frames that begin with an initiation codon, usually AUG,and lie between the ribosome binding site and the initiation codon.Also, generally, there will be a translation stop codon at the end ofthe polypeptide and there will be a polyadenylation signal in constructsfor use in eukaryotic hosts. Transcription termination signalsappropriately disposed at the 3′ end of the transcribed region may alsobe included in the polynucleotide construct.

For nucleic acid constructs designed to express a polypeptide, theexpression cassettes can additionally contain 5′ leader sequences. Suchleader sequences can act to enhance translation. Translation leaders areknown in the art and include: picornavirus leaders, for example: EMCVleader (Encephalomyocarditis 5′ noncoding region), Elroy-Stein et al.(1989) Proc. Nat. Acad. Sci. USA 86:6126-6130; potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMVleader (Maize Dwarf Mosaic Virus), Virology 154:9-20; humanimmunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991)Nature 353:90-94; untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV), Gallie et al. (1989)Molecular Biology of RNA, pages 237-256; and maize chlorotic mottlevirus leader (MCMV) Lommel et al. (1991) Virology 81:382-385. See alsoDella-Cioppa et al. (1987) Plant Physiology 84:965-968. The cassette canalso contain sequences that enhance translation and/or mRNA stabilitysuch as introns. The expression cassette also typically includes, at the3′ terminus of the isolated nucleotide sequence of interest, atranslational termination region, e.g., one functional in plants.

In those instances where it is desirable to have the expressed productof the isolated nucleotide sequence directed to a particular organelle,particularly the plastid, amyloplast, or to the endoplasmic reticulum,or secreted at the cell's surface or extracellularly, the expressioncassette can further comprise a coding sequence for a transit peptide.Such transit peptides are well known in the art and include, but are notlimited to: the transit peptide for the acyl carrier protein, the smallsubunit of RUBISCO, plant EPSP synthase, and the like.

In preparing the expression cassette, the various DNA fragments can bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers can be employed to join the DNA fragmentsor other manipulations can be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction digests, annealing, and resubstitutions such astransitions and transversions, can be involved.

As noted herein, the present invention provides vectors capable ofexpressing genes of interest under the control of the regulatoryelements. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional inother host cells, e.g., in E. coli (e.g., for production of protein forraising antibodies, DNA sequence analysis, construction of inserts, orobtaining quantities of nucleic acids). Vectors and procedures forcloning and expression in E. coli are discussed in Sambrook et al.(supra).

The transformation vector, comprising the promoter of the presentinvention operably linked to an isolated nucleotide sequence in anexpression cassette, can also contain at least one additional nucleotidesequence for a gene to be co-transformed into the organism.Alternatively, the additional sequence(s) can be provided on anothertransformation vector.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well-known techniques suitable to expressiontherein of a desired RNA and/or polypeptide. The present invention alsorelates to host cells containing the above-described constructs. Thehost cell can be a higher eukaryotic cell, such as a plant cell, or alower eukaryotic cell, such as a yeast cell, or the host cell can be aprokaryotic cell, such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS and Bowes melanoma cells; and plantcells. The plant cells may be derived from a broad range of plant types,particularly monocots such as the species of the Family Graminiaeincluding Sorghum bicolor and Zea mays, as well as dicots such assoybean (Glycine max) and canola (Brassica napus, Brassica rapa ssp.).Preferably, plants include maize, soybean, sunflower, safflower, canola,wheat, barley, rye, alfalfa, rice, oat, lawn grass, and sorghum;however, the isolated nucleic acid and proteins of the present inventioncan be used in species from the genera: Ananas, Antirrhinum,Arabidopsis, Arachis, Asparagus, Atropa, Avena, Brassica, Bromus,Browaalia, Camellia, Capsicum, Ciahorium, Citrus, Cocos, Cofea, Cucumis,Cucurbita, Datura, Daucus, Digitalis, Ficus, Fragaria, Geranium,Glycine,Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Ipomoea,Juglans, Lactuca, Linum, Lolium, Lotus, Lycopersicon, Majorana,Mangifera, Manihot, Medicago, Musa, Nemesis, Nicotiana, Olea,Onobrychis, Oryza, Panieum, Pelargonium, Pennisetum, Persea, Petunia,Phaseolus, Pisum, Psidium, Ranunculus, Raphanus, Rosa, Salpiglossis,Secale, Senecio, Solanum, Sinapis, Sorghum, Theobroma, Triticum,Trifolium, Trigonella, Vigna, Vitis, and Zea, among many other examples(e.g., other genera noted herein).

The promoter regions of the invention may be isolated from any plant,including, but not limited to, maize (corn; Zea mays), canola (Brassicanapus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryzasativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), oats, barley, vegetables, ornamentals, and conifers.Preferably, plants include maize, soybean, sunflower, safflower, canola,wheat, barley, rye, alfalfa, rice, oat, lawn grass, and sorghum.

Hosts for a great variety of expression constructs are well known, andthose of skill will be enabled by the present disclosure readily toselect a host for expressing a polypeptide in accordance with thisaspect of the present invention.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression generally will be suitable forexpression of nucleic acids and/or polypeptides of the presentinvention, as will be apparent to those of skill in the art.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, where the selected promoteris inducible it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period.

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents; such methods are well known to those skilled in the art.

Methods of Inhibiting Ethylene Production

The invention also provides methods for inhibiting ethylene productionin a plant (and plants produced by such methods). For example, a methodof inhibiting ethylene production comprises inactivating one or more ACCsynthase genes in the plant, wherein the one or more ACC synthase genesencodes one or more ACC synthases. Typically, at least one of the one ormore ACC synthases comprises, e.g., at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 99.5% or more identity to SEQID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO:9 (pAC7) or SEQ ID NO:11(pCCRA178R).

Antisense, Sense, RNA Silencing or Interference Configurations

The one or more ACC synthase gene can be inactivated by introducing andexpressing transgenic sequences, e.g., antisense or senseconfigurations, or RNA silencing or interference configurations, etc,encoding one or more ACC synthases, or a subsequence thereof, and apromoter, thereby inactivating the one or more ACC synthase genescompared to a corresponding control plant (e.g., its non-transgenicparent or a non-transgenic plant of the same species). See also thesection entitled “Polynucleotides of the Invention.” The at least onepolynucleotide sequence can be introduced by techniques including, butnot limited to, e.g., electroporation, micro-projectile bombardment,Agrobacterium-mediated transfer, or other available methods. See also,the section entitled “Plant Transformation,” herein. In certain aspectsof the invention, the polynucleotide is linked to the promoter in asense orientation or in an antisense orientation or is configured forRNA silencing or interference.

In certain situations it may be preferable to silence or down-regulatecertain genes, such as ACC synthase genes. Relevant literaturedescribing the application of homology-dependent gene silencingincludes: Jorgensen, Trends Biotechnol. 8 (12):340-344 (1990); Flavell,Proc. Nat'l. Acad. Sci. (USA) 91:3490-3496 (1994); Finnegan et al.,Bio/Technology 12:883-888 (1994); Neuhuber et al., Mol. Gen. Genet.244:230-241 (1994); Flavell et al. (1994) Proc. Natl. Acad. Sci. USA91:3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31:957-973;Johansen and Carrington (2001) Plant Physiol. 126:930-938; Broin et al.(2002) Plant Cell 14:1417-1432; Stoutjesdijk et al. (2002) PlantPhysiol. 129:1723-1731; Yu et al. (2003) Phytochemistry 63:753-763; andU.S. Pat. Nos. 5,034,323, 5,283,184, and 5,942,657. Alternatively,another approach to gene silencing can be with the use of antisensetechnology (Rothstein et al. in Plant Mol. Cell. Biol. 6:221-246 (1989);Liu et al. (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos.5,759,829 and 5,942,657.

Use of antisense nucleic acids is well known in the art. An antisensenucleic acid has a region of complementarity to a target nucleic acid,e.g., an ACC synthase gene, mRNA, or cDNA. The antisense nucleic acidcan be RNA, DNA, a PNA or any other appropriate molecule. A duplex canform between the antisense sequence and its complementary sensesequence, resulting in inactivation of the gene. The antisense nucleicacid can inhibit gene expression by forming a duplex with an RNAtranscribed from the gene, by forming a triplex with duplex DNA, etc. Anantisense nucleic acid can be produced, e.g., for an ACC synthase geneby a number of well-established techniques (e.g., chemical synthesis ofan antisense RNA or oligonucleotide (optionally including modifiednucleotides and/or linkages that increase resistance to degradation orimprove cellular uptake) or in vitro transcription). Antisense nucleicacids and their use are described, e.g., in U.S. Pat. No. 6,242,258 toHaselton and Alexander (Jun. 5, 2001) entitled “Methods for theselective regulation of DNA and RNA transcription and translation byphotoactivation”; U.S. Pat. No. 6,500,615; U.S. Pat. No. 6,498,035; U.S.Pat. No. 6,395,544; U.S. Pat. No. 5,563,050; E. Schuch et al (1991) SympSoc. Exp Biol 45:117-127; de Lange et al., (1995) Curr Top MicrobiolImmunol 197:57-75; Hamilton et al. (1995) Curr Top Microbiol Immunol197:77-89; Finnegan et al., (1996) Proc Natl Acad Sci USA 93:8449-8454;Uhlmann and A. Pepan (1990), Chem. Rev. 90:543; P. D. Cook (1991),Anti-Cancer Drug Design 6:585; J. Goodchild, Bioconjugate Chem. 1 (1990)165; and, S. L. Beaucage and R. P. Iyer (1993), Tetrahedron 49:6123; andF. Eckstein, Ed. (1991), Oligonucleotides and Analogues—A PracticalApproach, IRL Press.

Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of ACC synthase genes. It is possible to design ribozymesthat specifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules. The inclusion of ribozyme sequences within antisenseRNAs confers RNA-cleaving activity upon them, thereby increasing theactivity of the constructs.

A number of classes of ribozymes have been identified. For example, oneclass of ribozymes is derived from a number of small circular RNAs thatare capable of self-cleavage and replication in plants. The RNAs canreplicate either alone (viroid RNAs) or with a helper virus (satelliteRNAs). Examples of RNAs include RNAs from avocado sunblotch viroid andthe satellite RNAs from tobacco ringspot virus, lucerne transient streakvirus, velvet tobacco mottle virus, solanum nodiflorum mottle virus andsubterranean clover mottle virus. The design and use of targetRNA-specific ribozymes has been described. See, e.g., Haseloff et al.(1988) Nature, 334:585-591.

Another method to inactivate an ACC synthase gene by inhibitingexpression is by sense suppression. Introduction of expression cassettesin which a nucleic acid is configured in the sense orientation withrespect to the promoter has been shown to be an effective means by whichto block the transcription of a desired target gene. See, e.g., Napoliet al. (1990), The Plant Cell 2:279-289, and U.S. Pat. Nos. 5,034,323,5,231,020, and 5,283,184.

Isolated or recombinant plants which include one or more inactivated ACCsynthase gene can also be produced by using RNA silencing orinterference (RNAi), which can also be termed post-transcriptional genesilencing (PTGS) or cosuppression. In the context of this invention,“RNA silencing” (also called RNAi or RNA-mediated interference) refersto any mechanism through which the presence of a single-stranded or,typically, a double-stranded RNA in a cell results in inhibition ofexpression of a target gene comprising a sequence identical or nearlyidentical to that of the RNA, including, but not limited to, RNAinterference, repression of translation of a target mRNA transcribedfrom the target gene without alteration of the mRNA's stability, andtranscriptional silencing (e.g., histone acetylation and heterochromatinformation leading to inhibition of transcription of the target mRNA). In“RNA interference,” the presence of the single-stranded ordouble-stranded RNA in the cell leads to endonucleolytic cleavage andthen degradation of the target mRNA.

In one embodiment, a transgene (e.g., a sequence and/or subsequence ofan ACC synthase gene or coding sequence) is introduced into a plant cellto inactivate one or more ACC synthase genes by RNA silencing orinterference (RNAi). For example, a sequence or subsequence includes asmall subsequence, e.g., about 21-25 bases in length (with, e.g., atleast 80%, at least 90%, or about 100% identity to one or more of theACC synthase gene subsequences), a larger subsequence, e.g., about25-100 or about 100-2000 (or about 200-1500, about 250-1000, etc.) basesin length (with at least one region of about 21-25 bases of at least80%, at least 90%, or 100% identity to one or more ACC synthase genesubsequences), and/or the entire coding sequence or gene. In oneembodiment, a transgene includes a region in the sequence or subsequencethat is about 21-25 bases in length with at least 80%, at least 90%, orabout 100% identity to the ACC synthase gene or coding sequence.

Use of RNAi for inhibiting gene expression in a number of cell types(including, e.g., plant cells) and organisms, e.g., by expression of ahairpin (stem-loop) RNA or of the two strands of an interfering RNA, forexample, is well described in the literature, as are methods fordetermining appropriate interfering RNA(s) to target a desired gene,e.g., an ACC synthase gene, and for generating such interfering RNAs.For example, RNA interference is described e.g., in U.S. patentapplication publications 20020173478, 20020162126, and 20020182223 andin Cogoni and Macino (2000) “Post-transcriptionnal gene silencing acrosskingdoms” Genes Dev., 10:638-643; Guru T. (2000), “A silence that speaksvolumes” Nature 404: 804-808; Hammond et al., (2001),“Post-transcriptional Gene Silencing by Double-stranded RNA” Nature Rev.Gen. 2:110-119; Napoli et al., (1990) “Introduction of a chalconesynthase gene into Petunia results in reversible co-suppression ofhomologous genes in trans.” Plant Cell 2:279-289; Jorgensen et al.,(1996), “Chalcone synthase cosuppression phenotypes in petunia flowers:comparison of sense vs. antisense constructs and single-copy vs. complexT-DNA sequences” Plant Mol. Biol., 31:957-973; Hannon G. J. (2002) “RNAinterference” Nature., Jul 11; 418(6894):244-51; Ueda R. (2001) “RNAi: anew technology in the post-genomic sequencing era” J Neurogenet.;15(3-4):193-204; Ullu et al (2002) “RNA interference: advances andquestions” Philos Trans R Soc Lond B Biol Sci. Jan 29;357(1417):65-70;Waterhouse et al., (1998) Proc Natl Acad Sci USA 95:133959-13964;Schmidet al (2002) “Combinatorial RNAi: a method for evaluating the functionsof gene families in Drosophila” Trends Neurosci. Feb;25(2):71-4; Zeng etal. (2003) “MicroRNAs and small interfering RNAs can inhibit mRNAexpression by similar mechanisms” Proc. Natl. Acad. Sci. USA100:9779-9784; Doench et al. (2003) “siRNAs can function as miRNAs”Genes & Dev. 17:438-442; Bartel and Bartel (2003) “MicroRNAs: At theroot of plant development?” Plant Physiology 132:709-717; Schwarz andZamore (2002) “Why do miRNAs live in the miRNP?” Genes & Dev.16:1025-1031; Tang et al. (2003) “A biochemical framework for RNAsilencing in plants” Genes & Dev. 17:49-63; Meister et al. (2004)“Sequence-specific inhibition of microRNA- and siRNA-induced RNAsilencing” RNA 10:544-550; Nelson et al. (2003) “The microRNA world:Small is mighty” Trends Biochem. Sci. 28:534-540; Dykxhoorn et al.(2003) “Killing the messenger: Short RNAs that silence gene expression”Nature Reviews Molec. and Cell Biol. 4:457-467; McManus and Sharp (2002)“Gene silencing in mammals by small interfering RNAs” Nature ReviewsGenetics 3:737-747; Hutvagner and Zamore (2002) “RNAi: Nature abhors adouble strand” Curr Opin Genet & Dev 200:225-232; and Agami (2002) “RNAiand related mechanisms and their potential use for therapy” Curr OpinChem Biol 6:829-834.

The ACC synthase polynucleotide sequence(s) or subsequence(s) expressedto induce RNAi can be expressed, e.g., under control of a constitutivepromoter, an inducible promoter, or a tissue specific promoter.Expression from a tissue-specific promoter can be advantageous incertain embodiments. For example, expression from a leaf-specificpromoter can inactivate one or more ACC synthase genes in the leaf,producing a staygreen phenotype, without inactivating ACC synthase genesin the root (which can decrease flood tolerance). Similarly, expressionfrom an anther-specific promoter can inactivate one or more ACC synthasegenes in the anther, producing a male sterility phenotype, withoutinactivating ACC synthase genes in the remainder of the plant. Suchapproaches are optionally combined, e.g., to inactivate one or more ACCsynthase genes in both leaves and anthers.

Transposons

The one or more ACC synthase genes can also be inactivated by, e.g.,transposon based gene inactivation. In one embodiment, the inactivatingstep comprises producing one or more mutations in an ACC synthase genesequence, where the one or more mutations in the ACC synthase genesequence comprise one or more transposon insertions, therebyinactivating the one or more ACC synthase gene compared to acorresponding control plant. For example, the one or more mutationscomprise a homozygous disruption in the one or more ACC synthase gene orthe one or more mutations comprise a heterozygous disruption in the oneor more ACC synthase gene or a combination of both homozygousdisruptions and heterozygous disruptions if more than one ACC synthasegene is disrupted.

Transposons were first identified in maize by Barbara McClintock in thelate 1940s. The Mutator family of transposable elements, e.g.,Robertson's Mutator (Mu) transposable elements, are typically used inplant, e.g., maize, gene mutagenesis, because they are present in highcopy number (10-100) and insert preferentially within and around genes.

Transposable elements can be categorized into two broad classes based ontheir mode of transposition. These are designated Class I and Class II;both have applications as mutagens and as delivery vectors. Class Itransposable elements transpose by an RNA intermediate and use reversetranscriptases, i.e., they are retroelements. There are at least threetypes of Class I transposable elements, e.g., retrotransposons,retroposons, SINE-like elements.

Retrotransposons typically contain LTRs, and genes encoding viral coatproteins (gag) and reverse transcriptase, RnaseH, integrase andpolymerase (pol) genes. Numerous retrotransposons have been described inplant species. Such retrotransposons mobilize and translocate via a RNAintermediate in a reaction catalyzed by reverse transcriptase and RNaseH encoded by the transposon. Examples fall into the Ty1-copia andTy3-gypsy groups as well as into the SINE-like and LINE-likeclassifications. A more detailed discussion can be found in Kumar andBennetzen (1999) Plant Retrotransposons in Annual Review of Genetics33:479. In addition, DNA transposable elements such as Ac, Tam1 andEn/Spm are also found in a wide variety of plant species, and can beutilized in the invention.

Transposons (and IS elements) are common tools for introducing mutationsin plant cells. These mobile genetic elements are delivered to cells,e.g., through a sexual cross, transposition is selected for and theresulting insertion mutants are screened, e.g., for a phenotype ofinterest. Plants comprising disrupted ACC synthase genes can beintroduced into other plants by crossing the isolated or recombinantplants with a non-disrupted plant, e.g., by a sexual cross. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed. The location of a TN within a genome of anisolated or recombinant plant can be determined by known methods, e.g.,sequencing of flanking regions as described herein. For example, a PCRreaction from the plant can be used to amplify the sequence, which canthen be diagnostically sequenced to confirm its origin. Optionally, theinsertion mutants are screened for a desired phenotype, such as theinhibition of expression or activity of ACC synthase, inhibition orreduced production of ethylene, staygreen potential, etc. compared to acontrol plant.

Tilling

TILLING can also be used to inactivate one or more ACC synthase gene.TILLING is Targeting Induced Local Lesions IN Genomics. See, e.g.,McCallum et al., (2000), “Targeting Induced Local Lesions IN Genomics(TILLING) for Plant Functional Genomics” Plant Physiology 123:439-442;McCallum et al., (2000) “Targeted screening for induced mutations”Nature Biotechnology 18:455-457; and, Colbert et al., (2001)“High-Throughput Screening for Induced Point Mutations” Plant Physiology126:480-484.

TILLING combines high density point mutations with rapid sensitivedetection of the mutations. Typically, ethylmethanesulfonate (EMS) isused to mutagenize plant seed. EMS alkylates guanine, which typicallyleads to mispairing. For example, seeds are soaked in an about 10-20 mMsolution of EMS for about 10 to 20 hours; the seeds are washed and thensown. The plants of this generation are known as M1. M1 plants are thenself-fertilized. Mutations that are present in cells that form thereproductive tissues are inherited by the next generation (M2).Typically, M2 plants are screened for mutation in the desired geneand/or for specific phenotypes.

For example, DNA from M2 plants is pooled and mutations in an ACCsynthase gene are detected by detection of heteroduplex formation.Typically, DNA is prepared from each M2 plant and pooled. The desiredACC synthase gene is amplified by PCR. The pooled sample is thendenatured and annealed to allow formation of heteroduplexes. If amutation is present in one of the plants; the PCR products will be oftwo types: wild-type and mutant. Pools that include the heteroduplexesare identified by separating the PCR reaction, e.g., by Denaturing HighPerformance Liquid Chromatography (DPHPLC). DPHPLC detects mismatches inheteroduplexes created by melting and annealing of heteroallelic DNA.Chromatography is performed while heating the DNA. Heteroduplexes havelower thermal stability and form melting bubbles resulting in fastermovement in the chromatography column. When heteroduplexes are presentin addition to the expected homoduplexes, a double peak is seen. As aresult, the pools that carry the mutation in an ACC synthase gene areidentified. Individual DNA from plants that make up the selected pooledpopulation can then be identified and sequenced. Optionally, the plantpossessing a desired mutation in an ACC synthase can be crossed withother plants to remove background mutations.

Other mutagenic methods can also be employed to introduce mutations inan ACC synthase gene. Methods for introducing genetic mutations intoplant genes and selecting plants with desired traits are well known. Forinstance, seeds or other plant material can be treated with a mutagenicchemical substance, according to standard techniques. Such chemicalsubstances include, but are not limited to, the following: diethylsulfate, ethylene imine, and N-nitroso-N-ethylurea. Alternatively,ionizing radiation from sources such as X-rays or gamma rays can beused.

Other detection methods for detecting mutations in an ACC synthase genecan be employed, e.g., capillary electrophoresis (e.g., constantdenaturant capillary electrophoresis and single-stranded conformationalpolymorphism). In another example, heteroduplexes can be detected byusing mismatch repair enzymology (e.g., CEL I endonuclease from celery).CEL I recognizes a mismatch and cleaves exactly at the 3′ side of themismatch. The precise base position of the mismatch can be determined bycutting with the mismatch repair enzyme followed by, e.g., denaturinggel electrophoresis. See, e.g., Oleykowski et al., (1998) “Mutationdetection using a novel plant endonuclease” Nucleic Acid Res.26:4597-4602; and, Colbert et al., (2001) “High-Throughput Screening forInduced Point Mutations” Plant Physiology 126:480-484.

The plant containing the mutated ACC synthase gene can be crossed withother plants to introduce the mutation into another plant. This can bedone using standard breeding techniques.

Homologous Recombination

Homologous recombination can also be used to inactivate one or more ACCsynthase genes. Homologous recombination has been demonstrated inplants. See, e.g., Puchta et al. (1994), Experientia 50:277-284; Swobodaet al. (1994), EMBO J. 13:484-489; Offringa et al. (1993), Proc. Natl.Acad. Sci. USA 90:7346-7350; Kempin et al. (1997) Nature 389:802-803;and, Terada et al., (2002) “Efficient gene targeting by homologousrecombination in rice” Nature Biotechnology, 20(10):1030-1034.

Homologous recombination can be used to induce targeted genemodifications by specifically targeting an ACC synthase gene in vivo.Mutations in selected portions of an ACC synthase gene sequence(including 5′ upstream, 3′ downstream, and intragenic regions) such asthose provided herein are made in vitro and introduced into the desiredplant using standard techniques. The mutated gene will interact with thetarget ACC synthase wild-type gene in such a way that homologousrecombination and targeted replacement of the wild-type gene will occurin transgenic plants, resulting in suppression of ACC synthase activity.

Methods for Modulating Staygreen Potential in a Plant

Methods for modulating staygreen potential in plants are also featuresof the invention. The ability to introduce different degrees ofstaygreen potential into plants offers a flexible and simple approach tointroduce this trait in a purpose-specific manner: for example,introduction of a strong staygreen trait for improved grain-filling orfor silage in areas with longer or dryer growing seasons versus theintroduction of a moderate staygreen trait for silage in areas withshorter growing seasons. In addition, the staygreen potential of a plantof the invention can include, e.g., (a) a reduction in the production ofat least one ACC synthase specific mRNA; (b) a reduction in theproduction of an ACC synthase; (c) a reduction in the production ofethylene; (d) a delay in leaf senescence; (e) an increase of droughtresistance; (f) an increased time in maintaining photosyntheticactivity; (g) an increased transpiration; (h) an increased stomatalconductance; (i) an increased CO₂ assimilation; (j) an increasedmaintenance of CO₂ assimilation; or (k) any combination of (a)-(j);compared to a corresponding control plant, and the like.

For example, a method of the invention can include: a) selecting atleast one ACC synthase gene to mutate, thereby providing at least onedesired ACC synthase gene; b) introducing a mutant form of the at leastone desired ACC synthase gene into the plant; and, c) expressing themutant form, thereby modulating staygreen potential in the plant. Plantsproduced by such methods are also a feature of the invention.

The degree of staygreen potential introduced into a plant can bedetermined by a number of factors, e.g., which ACC synthase gene isselected, whether the mutant gene member is present in a heterozygous orhomozygous state, or by the number of members of this family which areinactivated, or by a combination of two or more such factors. In oneembodiment, selecting the at least one ACC synthase gene comprisesdetermining a degree (e.g., weak (e.g., ACS2), moderate or strong (e.g.,ACS6)) of staygreen potential desired. For example, ACS2 lines show aweak staygreen phenotype, with a delay in senescence of about 1 week.The ACS6 lines show a strong staygreen phenotype (e.g., leaf senescenceis delayed about 2-3 weeks or more). The ACS7 lines can also show astrong staygreen phenotype (e.g., leaf senescence is delayed about 2-3weeks or more). For example, the ACC synthase gene is selected forencoding a specific ACC synthase, such as, SEQ ID NO:7 (pACS2), SEQ IDNO:8 (pACS6), SEQ ID NO:9 (pAC7), or SEQ ID NO:11 (pCCRA178R). In oneembodiment, two or more ACC synthase genes are disrupted (e.g., ACS2 andACS6), e.g., to produce a strong staygreen phenotype. In otherembodiments, three or more ACC synthase genes are disrupted.

Once the desired ACC synthase gene is selected, a mutant form of the ACCsynthase gene is introduced into a plant. In certain embodiments, themutant form is introduced by Agrobacterium-mediated transfer,electroporation, micro-projectile bombardment, homologous recombinationor a sexual cross. In certain embodiments, the mutant form includes,e.g., a heterozygous mutation in the at least one ACC synthase gene, ahomozygous mutation in the at least one ACC synthase gene or acombination of homozygous mutation and heterozygous mutation if morethan one ACC synthase gene is selected. In another embodiment, themutant form includes a subsequence of the at least one desired ACCsynthase gene in an antisense, sense or RNA silencing or interferenceconfiguration.

Expression of the mutant form of the ACC synthase gene or result ofexpression of the mutant form can be determined in a number of ways. Forexample, detection of expression products is performed eitherqualitatively (presence or absence of one or more product of interest)or quantitatively (by monitoring the level of expression of one or moreproduct of interest). In one embodiment, the expression product is anRNA expression product. The invention optionally includes monitoring anexpression level of a nucleic acid or polypeptide as noted herein fordetection of ACC synthase in a plant or in a population of plants.Monitoring levels of ethylene or ACC can also be used for detection ofinhibition of expression or activity of a mutant form of the ACCsynthase gene.

In addition to increasing tolerance to drought stress in plants of theinvention compared to a control plant, another important aspect of theinvention is that higher density planting of plants of the invention canbe possible, leading to increased yield per acre of corn. Most of theincrease yield per acre of corn over the last century has come fromincreasing tolerance to crowding, which is a stress in, e.g., maize.Methods for modulating stress, e.g., increasing tolerance for crowding,in a plant are also a feature of the invention. For example, a method ofthe invention can include: a) selecting at least one ACC synthase geneto mutate, thereby providing at least one desired ACC synthase gene; b)introducing a mutant form of the at least one desired ACC synthase geneinto the plant; and, c) expressing the mutant form, thereby modulatingstress in the plant. Plants produced by such methods are also a featureof the invention. When the ethylene production is reduced in a plant bya mutant form of a desired ACC synthase gene, the plant does notperceive crowding. Thus, plants of the invention can be planted athigher density than currently practiced by farmers.

In another aspect, inactivation of one or more ACC synthase genes asdescribed herein can influence response to disease or pathogen attack.

Methods for Modulating Sterility in a Plant

Methods for modulating sterility, e.g., female or male sterility, inplants are also features of the invention. The ability to introducefemale or male sterility into plants permits rapid production of femaleor male sterile lines, e.g., for use in commercial breeding programs,e.g., for production of hybrid seed, where cross-pollination is desired.

ACC synthase knockout plants, particularly ACS6 knockouts and ACS2/ACS6double knockouts, have been observed to shed less pollen than wild-typeplants, suggesting disruption of ethylene production as a novel means ofmodulating plant sterility.

For example, a method of the invention can include: a) selecting atleast one ACC synthase gene to mutate, thereby providing at least onedesired ACC synthase gene; b) introducing a mutant form of the at leastone desired ACC synthase gene into the plant; and, c) expressing themutant form, thereby modulating sterility in the plant. Plants producedby such methods are also a feature of the invention.

Essentially all of the features noted above apply to this embodiment aswell, as relevant, for example, with respect to the number of ACCsynthase genes disrupted, techniques for introducing the mutant form ofthe ACC synthase gene into the plant, polynucleotide constructs, and thelike.

In one class of embodiments, the at least one ACC synthase gene isdisrupted by insertion of a transposon, by a point mutation, or byconstitutive expression of a transgene comprising an ACC synthasepolynucleotide in an antisense, sense, or RNA silencing or interferenceconfiguration. Such lines can be propagated by exogenously providingethylene, for example, by spraying the plants at an appropriatedevelopmental stage with 2-chloroethylphosphonic acid (CEPA), whichbreaks down in water to produce ethylene.

In another class of embodiments, the at least one ACC synthase gene isdisrupted by expression of a transgene comprising an ACC synthasepolynucleotide in an antisense, sense, or RNA silencing or interferenceconfiguration under the control of an inducible promoter, such thatsterility can be induced and/or repressed as desired. In yet anotherclass of embodiments, the at least one ACC synthase gene is disrupted byexpression of a transgene comprising an ACC synthase polynucleotide inan antisense, sense, or RNA silencing or interference configurationunder the control of a tissue-specific promoter, e.g., ananther-specific promoter to produce male sterile plants. Again, ifnecessary, such lines can be propagated by exogenously providingethylene (e.g., by spraying with CEPA.

Screening/Characterization of Plants or Plant Cells of the Invention

The plants of this invention can be screened and/or characterized eithergenotypically, biochemically, phenotypically or a combination of two ormore of the these to determine the presence, absence, and/or expression(e.g., amount, modulation, such as a decrease or increase compared to acontrol cell, and the like) of a polynucleotide of the invention, thepresence, absence, expression, and/or enzymatic activity of apolypeptide of the invention, modulation of staygreen potential,modulation of crowding, and/or modulation of ethylene production. See,e.g., FIG. 19.

Genotypic analysis can be performed by any of a number of well-knowntechniques, including PCR amplification of genomic nucleic acidsequences and hybridization of genomic nucleic acid sequences orexpressed nucleic acid sequences with specific labeled probes (e.g.,Southern blotting, northern blotting, dot or slot blots, etc.).

For example, the Trait Utility System for Corn (TUSC), developed byPioneer Hybrid Int., is a powerful PCR-based screening strategy toidentify Mu transposon insertions in specific genes without the need foran observable phenotype. The system utilizes, e.g., TIR-PCR in which onePCR primer is derived from the target gene and the other (Mu-TIR) fromthe terminal-inverted-repeat (TIR) region of Mu. Using these primers inPCR reactions of DNA pooled from a large population of Mu containingplants, successful amplification is identified by Southern hybridizationusing the target gene as the probe. Screening the individuals within apositive pool is then performed to identify the candidate linecontaining insertion of a Mu element in the target gene. In order todetermine whether an insertion event is limited to somatic cells or ispresent in the germ line (and therefore represents a heritable change),progeny from a candidate are optionally subjected to the samePCR/Southern hybridization analysis used in the original screen.

Biochemical analysis can also be performed for detecting, e.g., thepresence, the absence or modulation (e.g., decrease or increase) ofprotein production (e.g., by ELISAs, western blots, etc.), the presenceand/or amount of ethylene produced, and the like. For example, expressedpolypeptides can be recovered and purified from isolated or recombinantcell cultures by any of a number of methods well known in the art,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein. Finally, high performance liquid chromatography(HPLC) can be employed in the final purification steps. In addition tothe references noted above, a variety of purification methods are wellknown in the art, including, e.g., those set forth in Sandana (1997)Bioseparation of Proteins, Academic Press, Inc.; and Bollag et al.(1996) Protein Methods, 2^(nd) Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification: Principles and Practice 3^(rd) Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ.

Chemicals, e.g., ethylene, ACC, etc., can be recovered and assayed fromthe cell extracts. For example, internal concentrations of ACC can beassayed by gas chromatography-mass spectroscopy, in acidic plantextracts as ethylene after decomposition in alkaline hypochloritesolution, etc. The concentration of ethylene can be determined by, e.g.,gas chromatography-mass spectroscopy, etc. See, e.g., Nagahama, K.,Ogawa, T., Fujii, T., Tazaki, M., Tanase, S., Morino, Y. and Fukuda, H.(1991) “Purification and properties of an ethyleneforming enzyme fromPseudomonas syringae” J. Gen. Microbiol. 137:2281-2286. For example,ethylene can be measured with a gas chromatograph equipped with, e.g.,an alumina based column (such as an HP-PLOT A1203 capillary column) anda flame ionization detector.

Phenotypic analysis includes, e.g., analyzing changes in chemicalcomposition (e.g., as described under biochemical analysis), morphology,or physiological properties of the plant. For example, morphologicalchanges can include, but are not limited to, increased staygreenpotential, a delay in leaf senescence, an increase in droughtresistance, an increase in crowding resistance, etc. Physiologicalproperties can include, e.g., increased sustained photosynthesis,increased transpiration, increased stomatal conductance, increased CO₂assimilation, longer maintenance of CO₂ assimilation, etc.

A variety of assays can be used for monitoring staygreen potential. Forexample, assays include, but are not limited to, visual inspection,monitoring photosynthesis measurements, and measuring levels ofchlorophyll, DNA, RNA and/or protein content of, e.g., the leaves.

Plants of the Invention

Plant cells of the invention include, but are not limited to, meristemcells, Type I, Type II, and Type III callus, immature embryos, andgametic cells such as microspores, pollen, sperm and egg. In certainembodiments, the plant cell of the invention is from a dicot or monocot.A plant regenerated from the plant cell(s) of the invention is also afeature of the invention.

In one embodiment, the plant cell is in a plant, e.g., a hybrid plant,comprising a staygreen potential phenotype. In another embodiment, theplant cell is in a plant comprising a sterility phenotype, e.g., a malesterility phenotype. Through a series of breeding manipulations, thedisrupted ACC synthase gene can be moved from one plant line to anotherplant line. For example, the hybrid plant can be produced by sexualcross of a plant comprising a disruption in one or more ACC synthasegenes and a control plant.

Knockout plant cells are also a feature of the invention. In a firstaspect, the invention provides for an isolated or recombinant knockoutplant cell comprising at least one disruption in at least one endogenousACC synthase gene (e.g., a nucleic acid sequence, or complement thereof,comprising, e.g., at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 99%, about 99.5% or more, sequence identity to SEQ ID NO:1(gACS2), SEQ ID NO:2 (gACS6), or SEQ ID NO:3 (gACS7)). The disruptioninhibits expression or activity of at least one ACC synthase proteincompared to a corresponding control plant cell lacking the disruption.In one embodiment, the at least one endogenous ACC synthase genecomprises two or more endogenous ACC synthase genes. In anotherembodiment, the at least one endogenous ACC synthase gene comprisesthree or more endogenous ACC synthase genes. In certain embodiments, theat least one disruption results in reduced ethylene production by theknockout plant cell as compared to the control plant cell.

In one aspect of the invention, the disruption of an ACC synthase genein a plant cell comprises one or more transposons, wherein the one ormore transposons are in the at least one endogenous ACC synthase gene.In another aspect, the disruption includes one or more point mutationsin at least one endogenous ACC synthase gene. Optionally, the disruptionis a homozygous disruption in the at least one ACC synthase gene.Alternatively, the disruption is a heterozygous disruption in the atleast one ACC synthase gene. In certain embodiments, more than one ACCsynthase gene is involved and there is more than one disruption, whichcan include homozygous disruptions, heterozygous disruptions or acombination of homozygous disruptions and heterozygous disruptions. Seealso sections herein entitled “Transposons and TILLING.”

In another embodiment, the disruption of an ACC synthase gene isproduced by inhibiting expression of the ACC synthase gene. For example,a knockout plant cell is produced by introducing at least onepolynucleotide sequence comprising an ACC synthase nucleic acidsequence, or subsequence thereof, into a plant cell, such that the atleast one polynucleotide sequence is linked to a promoter in a sense orantisense orientation. The polynucleotide sequence comprises, e.g., atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about99.5% or more sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2(gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6),SEQ ID NO:6 (cACS7), or SEQ ID NO.:10 (CCRA178R), or a subsequencethereof, or a complement thereof. For example, the knockout plant cellcan be produced by introducing at least one polynucleotide sequencecomprising one or more subsequences of an ACC synthase nucleic acidsequence configured for RNA silencing or interference. Thepolynucleotide optionally comprises a vector, expression cassette, orthe like. In another aspect, the knockout plant cell is produced byhomologous recombination. See also sections herein entitled “Antisense,Sense, RNA Silencing or Interference Configurations” and “HomologousRecombination.”

Knockout plants that comprise a staygreen potential phenotype are afeature of the invention. Typically, the staygreen potential phenotypein the knockout plant results from a disruption in at least oneendogenous ACC synthase gene. In one embodiment, the disruptioncomprises one or more transposons, and the disruption inhibitsexpression or activity of at least one ACC synthase protein compared toa corresponding control plant. In another embodiment, the disruptioncomprises one or more point mutations and inhibits expression oractivity of the at least one ACC synthase protein compared to acorresponding control. In certain embodiments, the at least oneendogenous ACC synthase gene comprises a nucleic acid sequence, orcomplement thereof, comprising, e.g., at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 99.5% or more, sequenceidentity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), or SEQ ID NO:3(gACS7), or a complement thereof. In certain embodiments, the knockoutplant is a hybrid plant.

The invention also features knockout plants that comprise a transgenicplant with a staygreen potential phenotype. For example, a transgenicplant of the invention includes a staygreen potential phenotyperesulting from at least one introduced transgene which inhibits ethylenesynthesis, wherein said at least one introduced transgene comprises anucleic acid sequence encoding at least one ACC synthase or subsequencethereof, which nucleic acid sequence comprises, e.g., at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 99%, about 99.5% ormore, sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6(cACS7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or acomplement thereof, and modifies a level of expression or activity ofthe at least one ACC synthase. Typically, the configuration is a sense,antisense, or RNA silencing or interference configuration. A transgenicplant of the invention can also include a staygreen potential phenotyperesulting from at least one introduced transgene which inhibits ethylenesynthesis, wherein said at least one introduced transgene comprises anucleic acid sequence encoding subsequences of at least one ACCsynthase, which at least one ACC synthase comprises, e.g., at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, about 99.5%or more, sequence identity to SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6),SEQ ID NO.:9 (pACS7), or SEQ ID NO:11(pCCRA178R), or a conservativevariation thereof, and is in an RNA silencing or interferenceconfiguration, and modifies a level of expression or activity of the atleast one ACC synthase. In one aspect, the transgene optionallycomprises a tissue-specific promoter or an inducible promoter (e.g., aleaf-specific promoter, a drought-inducible promoter, or the like).

The invention also features knockout plants that have a sterilityphenotype, e.g., a male or female sterility phenotype. Thus, one classof embodiments provides a knockout plant comprising a male sterilityphenotype which results from at least one disruption in at least oneendogenous ACC synthase gene. The disruption inhibits expression oractivity of at least one ACC synthase protein compared to acorresponding control plant. For example, ACS2, ACS6, and ACS7 can bedisrupted, singly or in any combination (e.g., ACS6, or ACS2 and ACS6).Typically, the at least one disruption results in reduced ethyleneproduction by the knockout plant as compared to the control plant.

In one embodiment, the at least one disruption comprises one or moretransposons in the at least one endogenous ACC synthase gene. In anotherembodiment, the at least one disruption comprises one or more pointmutations in the at least one endogenous ACC synthase gene. In otherembodiments, the at least one disruption is introduced into the knockoutplant by introducing at least one polynucleotide sequence comprising oneor more subsequences of an ACC synthase nucleic acid sequence configuredfor RNA silencing or interference (or, alternatively, in a sense orantisense configuration). As noted, the polynucleotide sequence isoptionally under the control of an inducible or tissue-specific (e.g.,anther-specific) promoter.

In one embodiment, the male sterility phenotype comprises reduced pollenshedding by the knockout plant as compared to the control plant. Forexample, the knockout plant can shed at most 50%, 25%, 10%, 5%, or 1% asmuch pollen as the control plant, or it can shed no detectable pollen.

The invention also features knockout plants that comprise a transgenicplant with a male sterility phenotype. For example, a transgenic plantof the invention includes a male sterility phenotype resulting from atleast one introduced transgene which inhibits ethylene synthesis,wherein said at least one introduced transgene comprises a nucleic acidsequence encoding at least one ACC synthase or subsequence thereof,which nucleic acid sequence comprises, e.g., at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 99.5% or more,sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ IDNO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6(cACS7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or acomplement thereof, and modifies a level of expression or activity ofthe at least one ACC synthase. Typically, the configuration is a sense,antisense, or RNA silencing or interference configuration. As noted, thetransgene optionally comprises a tissue-specific promoter (e.g., ananther-specific promoter) or an inducible promoter.

Essentially any plant can be used in the methods and compositions of theinvention. Such species include, but are not restricted to members ofthe families: Poaceae (formerly Graminae, including Zea mays (corn),rye, triticale, barley, millet, rice, wheat, oats, etc.); Leguminosae(including pea, beans, lentil, peanut, yam bean, cowpeas, velvet beans,soybean, clover, alfalfa, lupine, vetch, lotus, sweet clover, wisteria,sweetpea, etc.); Compositae (the largest family of vascular plants,including at least 1,000 genera, including important commercial cropssuch as sunflower) and Rosaciae (including raspberry, apricot, almond,peach, rose, etc.), as well as nut plants (including, walnut, pecan,hazelnut, etc.), forest trees (including Pinus, Quercus, Pseutotsuga,Sequoia, Populus, etc.), and other common crop plants (e.g., cotton,sorghum, lawn grasses, tomato, potato, pepper, broccoli, cabbage, etc.)

Additional plants, as well as those specified above, include plants fromthe genera: Acamptoclados, Achnatherum, Achnella, Acroceras, Aegilops,Aegopgon, Agroelymus, Agrohordeum, Agropogon, Agropyron, Agrositanion,Agrostis, Aira, Allolepis, Alloteropsis, Alopecurus, Amblyopyrum,Ammophila, Ampelodesmos, Amphibromus, Amphicarpum, Amphilophis,Anastrophus, Anatherum, Andropogron, Anemathele, Aneurolepidium,Anisantha, Anthaenantia, Anthephora, Anthochloa, Anthoxanthum, Apera,Apluda, Archtagrostis, Arctophila, Argillochloa, Aristida,Arrhenatherum, Arthraxon, Arthrostylidium, Arundinaria, Arundinella,Arundo, Aspris, Atheropogon, Avena (e.g., oats), Avenella, Avenochloa,Avenula, Axonopus, Bambusa, Beckmannia, Blepharidachne, Blepharoneuron,Bothriochloa, Bouteloua, Brachiaria, Brachyelytrum, Brachypodium, Briza,Brizopyrum, Bromelica, Bromopsis, Bromus, Buchloe, Bulbilis,Calamagrostis, Calamovilfa, Campulosus, Capriola, Catabrosa, Catapodium,Cathestecum, Cenchropsis, Cenchrus, Centotheca, Ceratochloa,Chaetochloa, Chasmanthium, Chimonobambusa, Chionochloa, Chloris,Chondrosum, Chrysopon, Chusquea, Cinna, Cladoraphis, Coelorachis, Coix,Coleanthus, Colpodium, Coridochloa, Cornucopiae, Cortaderia,Corynephorus, Cottea, Critesion, Crypsis, Ctenium, Cutandia,Cylindropyrum, Cymbopogon, Cynodon, Cynosurus, Cytrococcum, Dactylis,Dactyloctenium, Danthonia, Dasyochloa, Dasyprum, Davyella,Dendrocalamus, Deschampsia, Desmazeria, Deyeuxia, Diarina, Diarrhena,Dichanthelium, Dichanthium, Dichelachne, Diectomus, Digitaria, Dimeria,Dimorpostachys, Dinebra, Diplachne, Dissanthelium, Dissochondrus,Distichlis, Drepanostachyum, Dupoa, Dupontia, Echinochloa, Ectosperma,Ehrharta, Eleusine, Elyhordeum, Elyleymus, Elymordeum, Elymus,Elyonurus, Elysitanion, Elytesion, Elytrigia, Enneapogon, Enteropogon,Epicampes, Eragrostis, Eremochloa, Eremopoa, Eremopyrum, Erianthus,Ericoma, Erichloa, Eriochrysis, Erioneuron, Euchlaena, Euclasta,Eulalia, Eulaliopsis, Eustachys, Fargesia, Festuca, Festulolium,Fingerhuthia, Fluminia, Garnotia, Gastridium, Gaudinia, Gigantochloa,Glyceria, Graphephorum, Gymnopogon, Gynerium, Hackelochloa, Hainardia,Hakonechloa, Haynaldia, Heleochloa, Helictotrichon, Hemarthria,Hesperochloa, Hesperostipa, Heteropogon, Hibanobambusa, Hierochloe,Hilaria, Holcus, Homalocenchrus, Hordeum (e.g., barley), Hydrochloa,Hymenachne, Hyparrhenia, Hypogynium, Hystrix, Ichnanthus, Imperata,Indocalamus, Isachne, Ischaemum, Ixophorus, Koeleria, Korycarpus,Lagurus, Lamarckia, Lasiacis, Leersia, Leptochloa, Leptochloopsis,Leptocoryphium, Leptoloma, Leptogon, Lepturus, Lerchenfeldia, Leucopoa,Leymostachys, Leymus, Limnodea, Lithachne, Lolium, Lophochlaena,Lophochloa, Lophopyrum, Ludolfia, Luziola, Lycurus, Lygeum, Maltea,Manisuris, Megastachya, Melica, Melinis, Mibora, Microchloa, Microlaena,Microstegium, Milium, Miscanthus, Mnesithea, Molinia, Monanthochloe,Monerma, Monroa, Muhlenbergia, Nardus, Nassella, Nazia, Neeragrostis,Neoschischkinia, Neostapfia, Neyraudia, Nothoholcus, Olyra, Opizia,Oplismenus, Orcuttia, Oryza (e.g., rice), Oryzopsis, Otatea,Oxytenanthera, Panicularia, Panicum, Pappophorum, Parapholis,Pascopyrum, Paspalidium, Paspalum, Pennisetum (e.g., millet), Phalaris,Phalaroides, Phanopyrum, Pharus, Phippsia, Phleum, Pholiurus,Phragmites, Phyllostachys, Piptatherum, Piptochaetium, Pleioblastus,Pleopogon, Pleuraphis, Pleuropogon, Poa, Podagrostis, Polypogon,Polytrias, Psathyrostachys, Pseudelymus, Pseudoroegneria, Pseudosasa,Ptilagrostis, Puccinellia, Pucciphippsia, Redfieldia, Reimaria,Reimarochloa, Rhaphis, Rhombolytrum, Rhynchelytrum, Roegneria,Rostraria, Rottboellia, Rytilix, Saccharum, Sacciolepis, Sasa, Sasaella,Sasamorpha, Savastana, Schedonnardus, Schismus, Schizachne,Schizachyrium, Schizostachyum, Sclerochloa, Scleropoa, Scleropogon,Scolochloa, Scribneria, Secale (e.g., rye), Semiarundinaria, Sesleria,Setaria, Shibataea, Sieglingia, Sinarundinaria, Sinobambusa,Sinocalamus, Sitanion, Sorghastrum, Sorghum, Spartina, Sphenopholis,Spodiopogon, Sporobolus, Stapfia, Steinchisma, Stenotaphrum, Stipa,Stipagrostis, Stiporyzopsis, Swallenia, Syntherisma, Taeniatherum,Terrellia, Terrelymus, Thamnocalamus, Themeda, Thinopyrum, Thuarea,Thysanolaena, Torresia, Torreyochloa, Trachynia, Trachypogon, Tragus,Trichachne, Trichloris, Tricholaena, Trichoneura, Tridens, Triodia,Triplasis, Tripogon, Tripsacum, Trisetobromus, Trisetum, Triticosecale,Triticum (e.g., wheat), Tuctoria, Uniola, Urachne, Uralepis, Urochloa,Vahlodea, Valota, Vaseyochloa, Ventenata, Vetiveria, Vilfa, Vulpia,Willkommia, Yushania, Zea (e.g., corn), Zizania, Zizaniopsis, andZoysia.

Plant Transformation

Nucleic acid sequence constructs of the invention (e.g., isolatednucleic acids, recombinant expression cassettes, etc.) can be introducedinto plant cells, either in culture or in the organs of plants, by avariety of conventional techniques. For example, techniques include, butare not limited to, infection, transduction, transfection, transvectionand transformation. The nucleic acid sequence constructs can beintroduced alone or with other polynucleotides. Such otherpolynucleotides can be introduced independently, co-introduced, orintroduced joined to polynucleotides of the invention.

Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Payne et al. (1992) Plant Cell and Tissue Culture in LiquidSystems John Wiley & Sons, Inc. New York, N.Y. (Payne); Gamborg andPhillips (eds) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg NewYork) (Gamborg); Croy, (ed.) (1993) Plant Molecular Biology BiosScientific Publishers, Oxford, U.K; Jones (ed) (1995) Plant GeneTransfer and Expression Protocols—Methods in Molecular Biology, Volume49 Humana Press Towata N.J., and as well as others, etc., as well as,e.g., Weising et al. (1988) Ann. Rev. Genet. 22:421. See, also, WO95/06128 entitled “Fertile, Transgenic Maize Plants and Methods forTheir Production” published on 2 Mar. 1995. Numerous protocols forestablishment of transformable protoplasts from a variety of plant typesand subsequent transformation of the cultured protoplasts are availablein the art and are incorporated herein by reference. For example, see,Hashimoto et al. (1990) Plant Physiol. 93:857; Fowke and Constabel (eds)(1994) Plant Protoplasts; Saunders et al. (1993) Applications of PlantIn Vitro Technology Symposium, UPM 16-18; and Lyznik et al. (1991)BioTechniques 10:295, each of which is incorporated herein by reference.Numerous methods are available in the art to accomplish chloroplasttransformation and expression (e.g., Daniell et al. (1998) NatureBiotechnology 16:346; O'Neill et al. (1993) The Plant Journal 3:729;Maliga (1993) TIBTECH 11:1).

For example, nucleic acid sequences can be introduced directly into thegenomic DNA of a plant cell using techniques such as electroporation,PEG poration, particle bombardment, silicon fiber delivery, ormicroinjection of plant cell protoplasts or embryogenic callus, or thenucleic acid sequence constructs can be introduced directly to planttissue using ballistic methods, such as particle bombardment. Exemplaryparticles include, but are not limited to, tungsten, gold, platinum, andthe like. Alternatively, the nucleic acid sequence constructs can beintroduced by infection of cells with viral vectors, or by combining thenucleic acid sequence constructs with suitable T-DNA flanking regionsand introduced into a conventional Agrobacterium tumefaciens hostvector. The virulence functions of the Agrobacterium host will directthe insertion of the construct and adjacent marker into the plant cellDNA when the plant cell is infected by the bacteria. See, U.S. Pat. No.5,591,616.

Microinjection techniques are known in the art and well described in thescientific and patent literature (see, e.g., Jones (ed) (1995) PlantGene Transfer and Expression Protocols—Methods in Molecular Biology,Volume 49 Humana Press Towata N.J., and as well as others). Theintroduction of nucleic acid sequence constructs using polyethyleneglycol precipitation is described in Paszkowski et al (1984) EMBO J3:2717. Electroporation techniques are described in Fromm et al. (1985)Proc Nat'l Acad Sci USA 82:5824. Ballistic transformation techniques aredescribed in Klein et al. (1987) Nature 327:70; and Weeks et al. PlantPhysiol 102:1077 and by Tomes, D. et al., IN: Plant Cell, Tissue andOrgan Culture: Fundamental Methods, Eds. O. L. Gamborg and G. C.Phillips, Chapter 8, pgs. 197-213 (1995). (See also Tomes et al., U.S.Pat. Nos. 5,886,244; 6,258,999; 6,570,067; 5,879,918).

Viral vectors which are plant viruses can also be used to introducepolynucleotides of the invention into plants. Viruses are typicallyuseful as vectors for expressing exogenous DNA sequences in a transientmanner in plant hosts. In contrast to agrobacterium mediatedtransformation which results in the stable integration of DNA sequencesin the plant genome, viral vectors are generally replicated andexpressed without the need for chromosomal integration. Plant virusvectors offer a number of advantages, specifically: a) DNA copies ofviral genomes can be readily manipulated in E.coli, and transcribed invitro, where necessary, to produce infectious RNA copies; b) naked DNA,RNA, or virus particles can be easily introduced into mechanicallywounded leaves of intact plants; c) high copy numbers of viral genomesper cell results in high expression levels of introduced genes; d)common laboratory plant species as well as monocot and dicot cropspecies are readily infected by various virus strains; e) infection ofwhole plants permits repeated tissue sampling of single library clones;f) recovery and purification of recombinant virus particles is simpleand rapid; and g) because replication occurs without chromosomalinsertion, expression is not subject to position effects. See, e.g.,Scholthof, Scholthof and Jackson, (1996) “Plant virus gene vectors fortransient expression of foreign proteins in plants,” Annu. Rev. ofPhytopathol. 34:299-323.

Plant viruses cause a range of diseases, most commonly mottled damage toleaves, so-called mosaics. Other symptoms include necrosis, deformation,outgrowths, and generalized yellowing or reddening of leaves. Plantviruses are known which infect every major food-crop, as well as mostspecies of horticultural interest. The host range varies betweenviruses, with some viruses infecting a broad host range (e.g., alfalfamosaic virus infects more than 400 species in 50 plant families) whileothers have a narrow host range, sometimes limited to a single species(e.g. barley yellow mosaic virus). Appropriate vectors can be selectedbased on the host used in the methods and compositions of the invention.

In certain embodiments of the invention, a vector includes a plantvirus, e.g., either RNA (single or double stranded) or DNA(single-stranded or doubled-stranded) virus. Examples of such virusesinclude, but are not limited to, e.g., an alfamovirus, a bromovirus, acapillovirus, a carlavirus, a carmovirus, a caulimovirus, aclosterovirus, a comovirus, a cryptovirus, a cucumovirus, adianthovirus, a fabavirus, a fijivirus, a furovirus, a geminivirus, ahordeivirus, a ilarvirus, a luteovirus, a machlovirus, a maize chloroticdwarf virus, a marafivirus, a necrovirus, a nepovirus, a parsnip yellowfleck virus, a pea enation mosaic virus, a potexvirus, a potyvirus, areovirus, a rhabdovirus, a sobemovirus, a tenuivirus, a tobamovirus, atobravirus, a tomato spotted wilt virus, a tombusvirus, a tymovirus, orthe like.

Typically, plant viruses encode multiple proteins required for initialinfection, replication and systemic spread, e.g. coat proteins, helperfactors, replicases, and movement proteins. The nucleotide sequencesencoding many of these proteins are matters of public knowledge, andaccessible through any of a number of databases, e.g. (Genbank:available on the World Wide Web at ncbi.nlm.nih.gov/genbank/ or EMBL:available on the World Wide Web at ebi.ac.uk.embl/).

Methods for the transformation of plants and plant cells using sequencesderived from plant viruses include the direct transformation techniquesdescribed above relating to DNA molecules, see e.g., Jones, ed. (1995)Plant Gene Transfer and Expression Protocols, Humana Press, Totowa, N.J.In addition, viral sequences can be cloned adjacent T-DNA bordersequences and introduced via Agrobacterium mediated transformation orAgroinfection.

Viral particles comprising the plant virus vectors includingpolynucleotides of the invention can also be introduced by mechanicalinoculation using techniques well known in the art; see, e.g.,Cunningham and Porter, eds. (1997) Methods in Biotechnology, Vol.3.Recombinant Proteins from Plants: Production and Isolation of ClinicallyUseful Compounds, for detailed protocols. Briefly, for experimentalpurposes, young plant leaves are dusted with silicon carbide(carborundum), then inoculated with a solution of viral transcript orencapsidated virus and gently rubbed. Large scale adaptations forinfecting crop plants are also well known in the art, and typicallyinvolve mechanical maceration of leaves using a mower or othermechanical implement, followed by localized spraying of viralsuspensions, or spraying leaves with a buffered virus/carborundumsuspension at high pressure. Any of these above mentioned techniques canbe adapted to the vectors of the invention, and are useful foralternative applications depending on the choice of plant virus and hostspecies, as well as the scale of the specific transformationapplication.

In some embodiments, Agrobacterium mediated transformation techniquesare used to transfer the ACC synthase sequences or subsequences of theinvention to transgenic plants. Agrobacterium-mediated transformation iswidely used for the transformation of dicots; however, certain monocotscan also be transformed by Agrobacterium. For example, Agrobacteriumtransformation of rice is described by Hiei et al. (1994) Plant J.6:271; U.S. Pat. No. 5,187,073; U.S. Pat. No. 5,591,616; Li et al.(1991) Science in China 34:54; and Raineri et al. (1990) Bio/Technology8:33. Transformed maize, barley, triticale and asparagus byAgrobacterium mediated transformation have also been described (Xu etal. (1990) Chinese J Bot 2:81).

Agrobacterium mediated transformation techniques take advantage of theability of the tumor-inducing (Ti) plasmid of A. tumefaciens tointegrate into a plant cell genome to co-transfer a nucleic acid ofinterest into a plant cell. Typically, an expression vector is producedwherein the nucleic acid of interest, such as an ACC synthase RNAconfiguration nucleic acid of the invention, is ligated into anautonomously replicating plasmid which also contains T-DNA sequences.T-DNA sequences typically flank the expression cassette nucleic acid ofinterest and comprise the integration sequences of the plasmid. Inaddition to the expression cassette, T-DNA also typically includes amarker sequence, e.g., antibiotic resistance genes. The plasmid with theT-DNA and the expression cassette are then transfected intoAgrobacterium cells. Typically, for effective transformation of plantcells, the A. tumefaciens bacterium also possesses the necessary virregions on a plasmid, or integrated into its chromosome. For adiscussion of Agrobacterium mediated transformation, see, Firoozabadyand Kuehnle, (1995) Plant Cell Tissue and Organ Culture FundamentalMethods, Gamborg and Phillips (eds.).

Agrobacterium tumefaciens-meditated transformation techniques are welldescribed in the scientific literature. See, for example Horsch et al.,Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci(USA) 80: 4803 (1983). Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include: (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, P W J Rigby,Ed., London, Academic Press, 1987; and Lichtenstein, C. P., and Draper,J,. In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press,1985); Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16, (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984),and (3) the vortexing method (see, e.g., Kindle, Proc. Nat'l. Acad.Sci.(USA) 87: 1228, (1990).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern. Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165(1988). Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena et al., Nature 325:274 (1987). DNA can alsobe injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings BioExpo. 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety ofplant viruses that can be employed as vectors are known in the art andinclude cauliflower mosaic virus (CaMV), geminivirus, brome mosaicvirus, and tobacco mosaic virus.

Other references describing suitable methods of transforming plant cellsinclude microinjection, Crossway et al. (1986) Biotechniques 4:320-334;electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA83:5602-5606; Agrobacterium-mediated transformation, see for example,Townsend et al. U.S. Pat. No. 5,563,055; direct gene transfer,Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particleacceleration, see for example, Sanford et al. U.S. Pat. No. 4,945,050;Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin);and McCabe et al. (1988) Biotechnology 6:923-926. Also see Weissinger etal. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987)Particulate Science and Technology 5:27-37 (onion); Christou et al.(1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839; Hooydaas-Van Slogteren et al. (1984)Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad.Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in TheExperimental Manipulation of Ovule Tissues, ed. G. P. Chapman et al.(Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant CellReports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D. Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou et al. (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

Regeneration of Isolated, Recombinant or Trangenic Plants

Transformed plant cells which are derived by plant transformationtechniques and isolated or recombinant plant cells, including thosediscussed above, can be cultured to regenerate a whole plant whichpossesses the desired genotype (i.e., a knockout ACC synthase nucleicacid), and/or thus the desired phenotype, e.g., staygreen phenotype,sterility phenotype, crowding resistant phenotype, etc. The desiredcells, which can be identified, e.g., by selection or screening, arecultured in medium that supports regeneration. The cells can then beallowed to mature into plants. For example, such regeneration techniquescan rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with the desired nucleotidesequences. Alternatively, screening can be performed to screen forinhibition of expression and/or activity of ACC synthase, reduction inethylene production conferred by the knockout ACC synthase nucleic acidsequence, etc. Plant regeneration from cultured protoplasts is describedin Evans et al. (1983) Protoplasts Isolation and Culture, Handbook ofPlant Cell Culture, pp 124-176, Macmillan Publishing Company, New York;Davey, (1983) Protoplasts, pp. 12-29, Birkhauser, Basal 1983; Dale,Protoplasts (1983) pp. 31-41, Birkhauser, Basel; and, Binding (1985)Regeneration of Plants, Plant Protoplasts pp 21-73, CRC Press, BocaRaton. Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann Rev of Plant Phys 38:467. See also,e.g., Payne and Gamborg. For transformation and regeneration of maizesee, for example, U.S. Pat. No. 5,736,369.

Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillilan Publishing Company, New York, pp. 124-176 (1983); andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

The regeneration of plants containing the foreign gene introduced byAgrobacterium from leaf explants can be achieved as described by Horschet al., Science, 227:1229-1231 (1985). After transformation withAgrobacterium, the explants typically are transferred to selectionmedium. One of skill will realize that the selection medium depends onthe selectable marker that is co-transfected into the explants. In thisprocedure, transformants are grown in the presence of a selection agentand in a medium that induces the regeneration of shoots in the plantspecies being transformed as described by Fraley et al., Proc. Nat'l.Acad. Sci. (U.S.A)., 80:4803 (1983). This procedure typically producesshoots, e.g., within two to four weeks, and these transformant shoots(which are typically, e.g., about 1-2 cm in length) are then transferredto an appropriate root-inducing medium containing the selective agentand an antibiotic to prevent bacterial growth. Selective pressure istypically maintained in the root and shoot medium.

Typically, the transformants will develop roots in about 1-2 weeks andform plantlets. After the plantlets are about 3-5 cm in height, they areplaced in sterile soil in fiber pots. Those of skill in the art willrealize that different acclimation procedures are used to obtaintransformed plants of different species. For example, after developing aroot and shoot, cuttings, as well as somatic embryos of transformedplants, are transferred to medium for establishment of plantlets. For adescription of selection and regeneration of transformed plants, see,e.g., Dodds and Roberts (1995) Experiments in Plant Tissue Culture,3^(rd) Ed., Cambridge University Press. Transgenic plants of the presentinvention may be fertile or sterile.

Regeneration can also be obtained from plant callus, explants, organs,or parts thereof. Such regeneration techniques are described generallyin Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). Theregeneration of plants from either single plant protoplasts or variousexplants is well known in the art. See, for example, Methods for PlantMolecular Biology, A. Weissbach and H. Weissbach, eds., Academic Press,Inc., San Diego, Calif. (1988). This regeneration and growth processincludes the steps of selection of transformant cells and shoots,rooting the transformant shoots and growth of the plantlets in soil. Formaize cell culture and regeneration see generally, The Maize Handbook,Freeling and Walbot, Eds., Springer, N.Y. (1994); Corn and CornImprovement, 3^(rd) edition, Sprague and Dudley Eds., American Societyof Agronomy, Madison, Wis. (1988).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed-propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype. Mature transgenic plants can also be crossed withother appropriate plants, generally another inbred or hybrid, including,for example, an isogenic untransformed inbred.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the isolated nucleicacid of the present invention. Progeny and variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these plants comprise the introduced nucleic acidsequences.

Transgenic plants expressing the selectable marker can be screened fortransmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then be analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

Some embodiments comprise a transgenic plant that is homozygous for theadded heterologous nucleic acid; i.e., a transgenic plant that containstwo added nucleic acid sequences, one gene at the same locus on eachchromosome of a chromosome pair. A homozygous transgenic plant can beobtained by sexually mating (selfing) a heterozygous (aka hemizygous)transgenic plant that contains a single added heterologous nucleic acid,germinating some of the seed produced and analyzing the resulting plantsproduced for altered expression of a polynucleotide of the presentinvention relative to a control plant (i.e., native, non-transgenic).Back-crossing to a parental plant and out-crossing with a non-transgenicplant, or with a plant transgenic for the same or another trait ortraits, are also contemplated.

It is also expected that the transformed plants will be used intraditional breeding programs, including TOPCROSS pollination systems asdisclosed in U.S. Pat. No. 5,706,603 and U.S. Pat. No. 5,704,160, thedisclosure of each of which is incorporated herein by reference.

In addition to Berger, Ausubel and Sambrook, useful general referencesfor plant cell cloning, culture and regeneration include Jones (ed)(1995) Plant Gene Transfer and Expression Protocols—Methods in MolecularBiology, Volume 49 Humana Press Towata N.J.; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y. (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) (Gamborg). A variety ofcell culture media are described in Atlas and Parks (eds) The Handbookof Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas).Additional information for plant cell culture is found in availablecommercial literature such as the Life Science Research Cell CultureCatalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC)and, e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc (St Louis, Mo.) (Sigma-PCCS). Additional detailsregarding plant cell culture are found in Croy, (ed.) (1993) PlantMolecular Biology Bios Scientific Publishers, Oxford, U.K.

“Stacking” of Constructs and Traits

In certain embodiments, the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The polynucleotides of the present invention may bestacked with any gene or combination of genes, and the combinationsgenerated can include multiple copies of any one or more of thepolynucleotides of interest. The desired combination may affect one ormore traits; that is, certain combinations may be created for modulationof gene expression affecting ACC synthase activity and/or ethyleneproduction. Other combinations may be designed to produce plants with avariety of desired traits, including but not limited to traits desirablefor animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529);balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389;5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson etal. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and highmethionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279;Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) PlantMol. Biol. 12:123)); increased digestibility (e.g., modified storageproteins (U.S. patent application Ser. No. 10/053,410, filed Nov. 7,2001); and thioredoxins (U.S. patent application Ser. No. 10/005,429,filed Dec. 3, 2001)), the disclosures of which are herein incorporatedby reference. The polynucleotides of the present invention can also bestacked with traits desirable for insect, disease or herbicideresistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos.5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881; Geiseret al (1986)Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones et al. (1994) Science 266:789; Martin etal. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089);acetolactate synthase (ALS) mutants that lead to herbicide resistancesuch as the S4 and/or Hra mutations; inhibitors of glutamine synthasesuch as phosphinothricin or basta (e.g., bar gene); and glyphosateresistance (EPSPS gene)); and traits desirable for processing or processproducts such as high oil (e.g., U.S. Pat. No. 6,232,529 ); modifiedoils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase),starch synthases (SS), starch branching enzymes (SBE) and starchdebranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S.Pat. No. 5.602,321; beta-ketothiolase, polyhydroxybutyrate synthase, andacetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol.170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)),the disclosures of which are herein incorporated by reference. One couldalso combine the polynucleotides of the present invention withpolynucleotides affecting agronomic traits such as male sterility (e.g.,see U.S. Pat. No. 5,583,210), stalk strength, flowering time, ortransformation technology traits such as cell cycle regulation or genetargeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosuresof which are herein incorporated by reference.

These stacked combinations can be created by any method, including butnot limited to cross breeding plants by any conventional or TopCrossmethodology, or genetic transformation. If the traits are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. For example, atransgenic plant comprising one or more desired traits can be used asthe target to introduce further traits by subsequent transformation. Thetraits can be introduced simultaneously in a co-transformation protocolwith the polynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences of interest can be driven bythe same promoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of a polynucleotide of interest. This may be accompanied byany combination of other suppression cassettes or over-expressioncassettes to generate the desired combination of traits in the plant.

Use in Breeding Methods

The transformed plants of the invention may be used in a plant breedingprogram. The goal of plant breeding is to combine, in a single varietyor hybrid, various desirable traits. For field crops, these traits mayinclude, for example, resistance to diseases and insects, tolerance toheat and drought, reduced time to crop maturity, greater yield, andbetter agronomic quality. With mechanical harvesting of many crops,uniformity of plant characteristics such as germination and standestablishment, growth rate, maturity, and plant and ear height isdesirable. Traditional plant breeding is an important tool in developingnew and improved commercial crops. This invention encompasses methodsfor producing a maize plant by crossing a first parent maize plant witha second parent maize plant wherein one or both of the parent maizeplants is a transformed plant displaying a staygreen phenotype, asterility phenotype, a crowding resistance phenotype, or the like, asdescribed herein.

Plant breeding techniques known in the art and used in a maize plantbreeding program include, but are not limited to, recurrent selection,bulk selection, mass selection, backcrossing, pedigree breeding, openpollination breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, doubled haploids, andtransformation. Often combinations of these techniques are used.

The development of maize hybrids in a maize plant breeding programrequires, in general, the development of homozygous inbred lines, thecrossing of these lines, and the evaluation of the crosses. There aremany analytical methods available to evaluate the result of a cross. Theoldest and most traditional method of analysis is the observation ofphenotypic traits. Alternatively, the genotype of a plant can beexamined.

A genetic trait which has been engineered into a particular maize plantusing transformation techniques can be moved into another line usingtraditional breeding techniques that are well known in the plantbreeding arts. For example, a backcrossing approach is commonly used tomove a transgene from a transformed maize plant to an elite inbred line,and the resulting progeny would then comprise the transgene(s). Also, ifan inbred line was used for the transformation, then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant. As used herein, “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, which, while different from each other, breedtrue and are highly uniform; and (3) crossing the selected inbred lineswith different inbred lines to produce the hybrids. During theinbreeding process in maize, the vigor of the lines decreases. Vigor isrestored when two different inbred lines are crossed to produce thehybrid. An important consequence of the homozygosity and homogeneity ofthe inbred lines is that the hybrid created by crossing a defined pairof inbreds will always be the same. Once the inbreds that give asuperior hybrid have been identified, the hybrid seed can be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

Transgenic plants of the present invention may be used to produce, e.g.,a single cross hybrid, a three-way hybrid or a double cross hybrid. Asingle cross hybrid is produced when two inbred lines are crossed toproduce the F1 progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F1 hybridsare crossed again (A×B)×(C×D). A three-way cross hybrid is produced fromthree inbred lines where two of the inbred lines are crossed (A×B) andthen the resulting F1 hybrid is crossed with the third inbred (A×B)×C.Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lostin the next generation (F2). Consequently, seed produced by hybrids isconsumed rather than planted.

Antibodies

The polypeptides of the invention can be used to produce antibodiesspecific for the polypeptides of SEQ ID NO:7-SEQ ID NO:9 and SEQ IDNO.:11, and conservative variants thereof. Antibodies specific for,e.g., SEQ ID NOs:7-9 and 11, and related variant polypeptides areuseful, e.g., for screening and identification purposes, e.g., relatedto the activity, distribution, and expression of ACC synthase.

Antibodies specific for the polypeptides of the invention can begenerated by methods well known in the art. Such antibodies can include,but are not limited to, polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments and fragments produced by an Fab expressionlibrary.

Polypeptides do not require biological activity for antibody production.The full length polypeptide, subsequences, fragments or oligopeptidescan be antigenic. Peptides used to induce specific antibodies typicallyhave an amino acid sequence of at least about 10 amino acids, and oftenat least 15 or 20 amino acids. Short stretches of a polypeptide, e.g.,selected from among SEQ ID NO:7-SEQ ID NO:9 and SEQ ID NO:11, can befused with another protein, such as keyhole limpet hemocyanin, andantibody produced against the chimeric molecule.

Numerous methods for producing polyclonal and monoclonal antibodies areknown to those of skill in the art and can be adapted to produceantibodies specific for the polypeptides of the invention, e.g.,corresponding to SEQ ID NO:7-SEQ ID NO:9 and SEQ ID NO:11. See, e.g.,Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Goding (1986) Monoclonal Antibodies: Principles andPractice (2d ed.) Academic Press, New York, N.Y.; FundamentalImmunology, e.g., 4^(th) Edition (or later), W. E. Paul (ed.), RavenPress, N.Y. (1998); and Kohler and Milstein (1975) Nature 256:495-497.Other suitable techniques for antibody preparation include selection oflibraries of recombinant antibodies in phage or similar vectors. See,Huse et al. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546. Specific monoclonal and polyclonal antibodies and antiserawill usually bind with a K_(D) of at least about 0.1 μM, preferably atleast about 0.01 μM or better, and most typically and preferably, 0.001μM or better.

Kits for Modulating Staygreen Potential or Sterility

Certain embodiments of the invention can optionally be provided to auser as a kit. For example, a kit of the invention can contain one ormore nucleic acid, polypeptide, antibody, diagnostic nucleic acid orpolypeptide, e.g., antibody, probe set, e.g., as a cDNA microarray, oneor more vector and/or cell line described herein. Most often, the kit ispackaged in a suitable container. The kit typically further comprisesone or more additional reagents, e.g., substrates, labels, primers, orthe like for labeling expression products, tubes and/or otheraccessories, reagents for collecting samples, buffers, hybridizationchambers, cover slips, etc. The kit optionally further comprises aninstruction set or user manual detailing preferred methods of using thekit components for discovery or application of gene sets. When usedaccording to the instructions, the kit can be used, e.g., for evaluatingexpression or polymorphisms in a plant sample, e.g., for evaluating ACCsynthase, ethylene production, staygreen potential, crowding resistancepotential, sterility, etc. Alternatively, the kit can be used accordingto instructions for using at least one ACC synthase polynucleotidesequence to control staygreen potential in a plant.

As another example, a kit for modulating sterility, e.g., malesterility, in a plant includes a container containing at least onepolynucleotide sequence comprising a nucleic acid sequence, wherein thenucleic acid sequence is, e.g., at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, about 99.5% or more, identical to SEQ IDNO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4(cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cAC7) or SEQ ID NO:10(CCRA178R), or a subsequence thereof, or a complement thereof. The kitoptionally also includes instructional materials for the use of the atleast one polynucleotide sequence to control sterility, e.g., malesterility, in a plant.

Other Nucleic Acid and Protein Assays

In the context of the invention, nucleic acids and/or proteins aremanipulated according to well known molecular biology methods. Detailedprotocols for numerous such procedures are described in, e.g., inAusubel et al. Current Protocols in Molecular Biology (supplementedthrough 2004) John Wiley & Sons, New York (“Ausubel”); Sambrook et al.Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989 (“Sambrook”), andBerger and Kimmel Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif.(“Berger”).

In addition to the above references, protocols for in vitroamplification techniques, such as the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA), useful e.g., foramplifying polynucleotides of the invention, are found in Mullis et al.(1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (“Innis”); Arnheim and Levinson (1990) C&EN 36; The Journal OfNIH Research (1991) 3:81; Kwoh et al. (1989) Proc Natl Acad Sci USA 86,1173; Guatelli et al. (1990) Proc Natl Acad Sci USA 87:1874; Lomell etal. (1989) J Clin Chem 35:1826; Landegren et al. (1988) Science241:1077; Van Brunt (1990) Biotechnology 8:291; Wu and Wallace (1989)Gene 4:560; Barringer et al. (1990) Gene 89:117, and Sooknanan and Malek(1995) Biotechnology 13:563. Additional methods, useful for cloningnucleic acids in the context of the invention, include Wallace et al.U.S. Pat. No. 5,426,039. Improved methods of amplifying large nucleicacids by PCR are summarized in Cheng et al. (1994) Nature 369:684 andthe references therein.

Certain polynucleotides of the invention can be synthesized utilizingvarious solid-phase strategies involving mononucleotide- and/ortrinucleotide-based phosphoramidite coupling chemistry. For example,nucleic acid sequences can be synthesized by the sequential addition ofactivated monomers and/or trimers to an elongating polynucleotide chain.See e.g., Caruthers, M. H. et al. (1992) Meth Enzymol 211:3. In lieu ofsynthesizing the desired sequences, essentially any nucleic acid can becustom ordered from any of a variety of commercial sources, such as TheMidland Certified Reagent Company (mcrc@oligos.com) (Midland, Tex.), TheGreat American Gene Company (available on the World Wide Web atgenco.com) (Ramona, Calif.), ExpressGen, Inc. (available on the WorldWide Web at expressgen.com) (Chicago Ill.), Operon Technologies, Inc.(available on the World Wide Web at operon.com) (Alameda Calif.), andmany others.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Isolation of Maize ACC Synthase Knockouts

Because ethylene has been associated with promoting leaf senescence insome species, to introduce staygreen potential into, e.g., maize, weundertook to reduce ethylene biosynthesis in maize leaves through theinactivation of ACC synthase genes. The maize ACC synthase gene familyis composed of three members: ACS2, ACS6, and ACS7. In order to isolateethylene mutants, we screened for disruptions of each member of the ACCsynthase gene family using the Trait Utility System for Corn (TUSC). Todate, we have determined the exact Mu insertion site for 8 mutant lines(three ACS6 and five ACS2) by sequencing across the Mu/ACC synthasejunction. Five insertions were stably inherited; their positions areindicated in FIG. 2, which also schematically depicts ACS7.

A pronounced staygreen phenotype was observed in leaves of those plantsin which a single gene member of the ACS family was present in aheterozygous mutant state (see FIG. 3, Panels A, B, C, and D). Whenpresent in a homozygous mutant state, an even more pronounced staygreenphenotype was observed (see FIG. 4). In FIG. 4, a leaf from thewild-type (left), heterozygous ACC synthase knockout (middle), andhomozygous ACC synthase knockout (right) was sheathed for seven (7) daysin the dark. Leaves from homozygous ACC synthase knockout plantsexhibited a greater staygreen trait than leaves of the heterozygous ACCsynthase knockout and exhibited a substantially greater staygreen traitthan leaves of wild type plants.

The degree of staygreen potential introduced was gene member specific.Consequently, a strong staygreen trait was introduced with the mutationof one member (e.g., ACS6), while a less pronounced staygreen phenotypewas introduced with the mutation of another member (e.g., ACS2).Therefore, the degree of staygreen potential introduced into a line canbe controlled by which mutant gene member is introduced, whether themutant gene member is present in a heterozygous or homozygous state, andby the number of members of this family which are inactivated (e.g.,ACS2/ACS6 double mutants have a strong staygreen phenotype). Traitsassociated with improved hybrid standability include resistance to stalkrot and leaf blights, genetic stalk strength, short plant height and earplacement, and high staygreen potential.

Typically, leaves follow a typical progression from initiation throughexpansion ultimately ending in senescence. The carbon fixation capacityalso increases during expansion and ultimately declines to low levelsthroughout senescence. See, e.g., Gay A P, and Thomas H (1995) Leafdevelopment in Lolium temulentum: photosynthesis in relation to growthand senescence. New Phytologist 130:159-168. This is of particularrelevance to cereal species where yield potential is largely dependentupon the ability of the plant to fix carbon and store this carbon in theseed, mainly in the form of starch. Both the timing at which senescenceis initiated and the rate at which it progresses can have a significantimpact on the overall carbon a particular leaf can ultimately contributeto a plant. See, e.g., Thomas H, and Howarth C J (2000) Five ways tostay green. Journal of Experimental Botany 51:329-337. This is ofparticular relevance to those crops where yield potential is reduced byadverse environmental conditions that induce premature leaf senescence.Stay-green is a general term used to describe a phenotype whereby leafsenescence (most easily distinguished by yellowing of the leafassociated with chlorophyll degradation) is delayed compared to astandard reference. See, e.g., Thomas and Howarth, supra. In sorghum,several stay-green genotypes have been identified which exhibit a delayin leaf senescence during grain filling and maturation. See, e.g.,Duncan R R, et al. (1981) Descriptive comparison of senescent andnon-senescent sorghum genotypes. Agronomy Journal 73:849-853. Moreover,under conditions of limited water availability, which normally hastensleaf senescence (e.g., Rosenow D T, and Clark L E (1981) Droughttolerance in sorghum. In: Loden H D, Wilkinson D, eds. Proceedings ofthe 36th annual corn and sorghum industry research conference, 18-31),these genotypes retain more green leaf area and continue to fill grainnormally (e.g., McBee G G, et al. (1983) Effect of senescence andnon-senescence on carbohydrates in sorghum during late kernel maturitystates. Crop Science 23:372-377; Rosenow D T, et al. (1983)Drought-tolerant sorghum and cotton germplasm. Agricultural WaterManagement 7:207-222; and, Borrell A K, Douglas A C L (1996) Maintaininggreen leaf area in grain sorghum increases yield in a water-limitedenvironment. In: Foale M A, Henzell R G, Kneipp J F, eds. Proceedings ofthe third Australian sorghum conference. Melbourne: Australian Instituteof Agricultural Science, Occasional Publication No. 93). The stay-greenphenotype has also been used as a selection criterion for thedevelopment of improved varieties of corn, particularly with regard tothe development of drought-tolerance. See, e.g., Russell W A (1991)Genetic improvement of maize yields. Advances in Agronomy 46:245-298;and, Bruce et al., (2002), Molecular and physiological approaches tomaize improvement for drought tolerance, Journal of Experimental Botany,53 (366):13-25.

Five fundamentally distinct types of stay-green have been described.See, e.g., Thomas H, and Smart C M (1993) Crops that stay green. Annalsof Applied Biology 123:193-219; and, Thomas and Howarth, supra. In TypeA stay-green, initiation of the senescence program is delayed, but thenproceeds at a normal rate. In Type B stay-green, while initiation of thesenescence program is unchanged, the progression is comparativelyslower. In Type C stay-green, chlorophyll is retained even thoughsenescence (as determined through measurements of physiological functionsuch as photosynthetic capacity) proceeds at a normal rate. Type Dstay-green is more artificial in that killing of the leaf (i.e. byfreezing, boiling or drying) prevents initiation of the senescenceprogram thereby stopping the degradation of chlorophyll. In Type Estay-green, initial levels of chlorophyll are higher while initiationand progression of leaf senescence are unchanged, thereby giving theillusion of a relatively slower progression rate. Type A and B arefunctional stay-greens as photosynthetic capacity is maintained alongwith chlorophyll content and are the types associated with increasedyield and drought tolerance in sorghum. Despite the potential importanceof this trait, in particular the benefits associated with increasingyield and drought tolerance, very little progress has been made inunderstanding the biochemical, physiological or molecular basis forgenetically-determined stay-green. See, e.g., Thomas and Howarth, supra.

A number of environmental and physiological conditions have been shownto significantly alter the timing and progression of leaf senescence andcan provide some insight into the basis for this trait. Amongenvironmental factors, light is probably the most significant and it haslong been established that leaf senescence can be induced in many plantspecies by placing detached leaves in darkness. See, e.g., Weaver L M,Amasino R M (2001) Senescence is induced in individually darkenedArabidopsis leaves, but inhibited in whole darkened plants. PlantPhysiology 127:876-886. Limited nutrient and water availability havealso been shown to induce leaf senescence prematurely (e.g., Rosenow DT, Quisenberry J E, Wendt C W, Clark L E (1983) Drought-tolerant sorghumand cotton germplasm. Agricultural Water Management 7:207-222). Amongphysiological determinants, growth regulators play a key role indirecting the leaf senescence program. Of particular relevance is theobservation that modification of cytokinin levels can significantlydelay leaf senescence. For example, plants transformed with isopentenyltransferase (ipt), an Agrobacterium gene encoding a rate-limiting stepin cytokinin biosynthesis, when placed under the control of a senescenceinducible promoter, resulted in autoregulated cytokinin production and astrong stay-green phenotype. See, e.g., Gan S, Amasino R M (1995)Inhibition of leaf senescence by autoregulated production of cytokinin.Science 270:1986-1988. Ethylene has also been implicated in controllingleaf senescence (e.g., Davis K M, and Grierson D (1989) Identificationof cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs thataccumulate during fruit ripening and leaf senescence in response toethylene. Planta 179:73-80) and plants impaired in ethylene productionor perception also show a delay in leaf senescence (e.g., Picton S, etal., (1993) Altered fruit ripening and leaf senescence in tomatoesexpressing an antisense ethylene-forming enzyme transgene. The PlantJournal 3:469-481; Grbic V, and Bleeker A B (1995) Ethylene regulatesthe timing of leaf senescence in Arabidopsis. The Plant Journal8:95-102; and, John I, et al., (1995) Delayed leaf senescence inethylene-deficient ACC-oxidase antisense tomato plants: molecular andphysiological analysis. The Plant Journal 7:483-490), which can bephenocopied by exogenous application of inhibitors of ethylenebiosynthesis and action (e.g., Abeles F B, et al., (1992) Ethylene inPlant Biology. Academic Press, San Diego, Calif.).

The identification and analysis of mutants in Arabidopsis and tomatothat are deficient in ethylene biosynthesis and perception are valuablein establishing the important role that ethylene plays in plant growthand development. Mutant analysis has also been instrumental inidentifying and characterizing the ethylene signal transduction pathway.While many ethylene mutants have been identified in dicot plants (e.g.,Arabidopsis and tomato), no such mutants have been identified inmonocots (e.g., rice, wheat, and corn). Here the identification of maizemutants deficient in ACC synthase, the first enzyme in the ethylenebiosynthetic pathway, are described. These mutants are critical inelucidating the regulatory roles that ethylene plays throughout cerealdevelopment as well as its role in regulating responses to environmentalstress. Knowledge obtained from such mutant analysis will increase theunderstanding of the role of ethylene in maize development and will bepertinent to other cereal crop species.

Mutants were deficient in ethylene production and exhibited a staygreenphenotype. Staygreen was observed under normal growth conditions andfollowing prolonged conditions of drought that induced premature onsetof leaf senescence in wild-type plants. In addition to the maintenanceof chlorophyll during water stress, ACC synthase-deficient leavesmaintained photosynthetic function and continued to assimilate CO₂.Surprisingly, reducing ethylene production improved leaf function in allleaves under normal growth conditions and maintained a high level offunction in drought-stressed plants even for those leaves in whichsenescence had not been induced in similar age leaves of wild-typeplants. These findings indicate that ethylene may serve to regulate leaffunction under normal growth conditions as well as in response toconditions of drought.

Materials and Methods

Cloning of ACC synthase genes from Zea mays

To facilitate cloning of ACC synthase gene(s) from maize, primers weredesigned to regions highly conserved between multiple monocot and dicotspecies using sequence information currently available in GenBank.Initial PCR reactions were carried out on maize genomic DNA usingprimers ACCF1 (ccagatgggcctcgccgagaac; SEQ ID NO:12) and ACC1(gttggcgtagcagacgcggaacca; SEQ ID NO:13) and revealed the presence ofthree fragments of different sizes. All three fragments were sequencedand confirmed to be highly similar in sequence to other known ACCsynthase genes.

To obtain the entire genomic sequences for each of these genes, allthree fragments were radiolabeled with dCTP using the Prime-a-Genelabeling system (Promega) and used to screen an EMBL3 maize (B73)genomic library (Stratagene) according to methods described in Sambrook,supra. Hybridization was carried out overnight at 30° C. in buffercontaining 5×SSPE, 5× Denhardt's, 50% Formamide and 1% SDS. Blots werewashed sequentially at 45° C. in 1×SSPE and 0.1×SSPE containing 0.1% SDSand exposed to film at −80° C. with an intensifier screen. A total of 36confluent plates (150 mm diameter) were screened. Putative positivesplaques were subsequently screened directly by PCR using the aboveprimers to identify which clones contained fragments corresponding tothe three fragments initially identified. PCR screening was accomplishedusing HotStarTaq (Qiagen). Reactions contained 1× buffer, 200 μM of eachdNTP, 3 μM MgCl₂, 0.25 μM forward and reverse primer, 1.25 U HotStarTaqand 1 μl primary phage dilution (1/600 total in SM buffer) as a templatein a total reaction volume of 25 μl. Reaction conditions were asfollows: 95° C./15 min. (1 cycle); 95° C./1 min, 62° C./1 min, 72° C./2min (35 cycles); 72° C./5 min (1 cycle). Samples were separated on a 1%agarose gel and the products were visualized following staining withethidium bromide. All fragments amplified were also subjected torestriction analysis to identify other potential sequence specificdifferences independent of subsequence size.

To facilitate sequencing the remaining portions of these genes, primersACCF1 and ACC1 were used in conjunction with primers specific to eitherthe left (gacaaactgcgcaactcgtgaaaggt; SEQ ID NO:14) or right(ctcgtccgagaataacgagtggatct; SEQ ID NO:15) arm of the EMBL3 vector toamplify each half of the gene. Takara LA Taq (Panvera) was used toamplify the fragments due to the large size. Reactions contained 1 μlphage dilution (1/600 total in SM buffer) and 2 μM each primer (finalconcentration), 1× buffer (final concentration), 400 μM dNTP mix (finalconcentration) and 1.25 U LA Taq in 25 μl total volume. Reactions werecarried out under the following conditions: 98° C./1 min. (1 cycle); 98°C./30 sec, 69° C./15 min. (35 cycles); 72° C./10 min (1 cycle).Amplified products were purified using the StrataPrep PCR purificationkit (Stratagene) and sent to the sequencing facility at the Universityof Florida, Gainesville for direct sequencing.

Identification of ACC Synthase Knock Out Mutants

Maize has proven to be a rich source of mutants, in part due to thepresence of active or previously active transposable element systemswithin its genome. Depending precisely on the location of the insertionsite in a gene, a transposon can partially or completely inactivateexpression of a gene. Gene inactivation may or may not have anobservable phenotype depending upon the amount of redundancy (i.e.presence of multiple family members and the tissue specificity of thefamily members). Trait Utility System for Corn (TUSC), developed byPioneer Hybrid Int., is a powerful PCR-based screening strategy toidentify Mu transposon insertions in specific genes without the need foran observable phenotype. This screening approach is best suited totarget genes that have been previously isolated from maize. The systemutilizes TIR-PCR in which one PCR primer is derived from the target geneand the other (Mu-TIR) from the terminal-inverted-repeat (TIR) region ofMu. Using these primers in PCR reactions of DNA pooled from a largepopulation of Mu containing plants, successful amplification isidentified by Southern hybridization using the target gene as the probe.Screening the individuals within a positive pool is then performed toidentify the candidate line containing insertion of a Mu element in thetarget gene. In order to determine whether an insertion event is limitedto somatic cells or is present in the germ line (and thereforerepresents a heritable change), progeny from a candidate are subjectedto the same PCR/Southern hybridization analysis used in the originalscreen.

A research effort was established to identify knockout mutants inethylene biosynthesis. To accomplish this, four primers (ACCF1,ccagatgggcctcgccgagaac, SEQ ID NO:12; ACC-1, gttggcgtagcagacgcggaacca,SEQ ID NO:13; ACC-C, cagttatgtgagggcacaccctacagcca, SEQ ID NO:16; ACC-D,catcgaatgccacagctcgaacaacttc, SEQ ID NO:17) specific to the maize ACCsynthase genes discussed above were used to screen for Mu insertions incombination with the Mu-TIR primer (aagccaacgcca(a/t)cgcctc(c/t)atttcgt;SEQ ID NO:18). The initial screening resulted in the putativeidentification of 19 separate lines carrying Mu insertions in the maizeACC synthase multigene family. Seed from each of these lines was plantedand DNA was extracted from the leaf of each individual. For DNAisolation, 1 cm² of seedling leaf was isolated from each plant andplaced into a 1.5 ml centrifuge tube containing some sand. Samples werequick-frozen in liquid nitrogen and ground to a fine powder using adisposable pestle (Fisher Scientific). 600 μl of extraction buffer (100mM Tris (pH 8.0), 50 mM EDTA, 200 mM NaCl, 1% SDS, 10 μl/mlβ-mercaptoethanol) was added immediately and mixed thoroughly. 700 μlPhenol/Chloroform (1:1) was added and samples were centrifuged 10 min at12,000 rpm. 500 μl supernatant was removed to a new tube and the nucleicacid precipitated at −20° C. following addition of 1/10 vol 3M sodiumacetate and 1 vol isopropanol. Total nucleic acid was pelleted bycentrifugation at 12,000 rpm, washed 3× with 75% ethanol and resuspendedin 600 μl H₂O. PCR screening was accomplished using HotStarTaq (Qiagen).Reactions contained 1× buffer, 200 μM of each dNTP, 3 mM MgCl2, 0.25 μMACC synthase specific primer (ACCF1, ACC-1, ACC-C or ACC-D), 0.25 μM Muspecific primer (MuTIR), 0.25 μl HotStarTaq and 1.5 μl total nucleicacid as a template in a total reaction volume of 25 μl. Reactionconditions were as follows: 95° C./15 min. (1 cycle); 95° C./1 min, 62°C./1 min, 72° C./2 min (35 cycles); 72° C./5 min (1 cycle). PCR productswere separated on a 1% agarose gel, visualized following staining withethidium bromide and transferred to nylon membranes according to methodsdescribed in Sambrook et al. (1989). Southern blot analysis wasperformed as described above for library screening except thehybridization temperature was increased to 45° C. BC1 (backcross 1) seedwas planted from each of the 13 putative mutant lines and screened byPCR/Southern analysis (as just described). Of these lines, only 5 werefound to be stably inherited. These five lines were backcrossed anadditional 4 times to minimize the effects of unrelated Mu insertions.

BC5 seed was self-pollinated to generate homozygous null individuals.Homozygous null individual lines were identified by PCR using Takara LATaq and the ACCF1 and ACC-1 primers. Reactions contained 1 μl leaf DNA,2 μM each primer (final concentration), 1× buffer (final concentration),400 μM dNTP mix (final concentration) and 1.25 U LA Taq in 25 μl totalvolume. Reactions were carried out under the following conditions: 98°C./1 min. (1 cycle); 98° C./30 sec, 69° C./15 min. (35 cycles); 72°C./10 min (1 cycle). PCR of wild-type B73 DNA using these primers andconditions results in the amplification of three different sizedfragments corresponding to the three genes identified. Individuals whichare either wild-type or heterozygous for one of the nullinsertion-alleles display this characteristic pattern while those whichare homozygous for one of the null insertion-alleles are missing thesubsequence corresponding to the gene in which the insertion is located.

To determine the exact location of the Mu insertion site, PCR productsfrom each of the lines were amplified using either the ACCF1 or ACC-1primer in combination with the MuTIR primer. These fragments were thensequenced across the Mu/target gene junction using the Mu-TIR primer.The location of these Mu elements within each of the ACC synthase genesis shown in FIG. 2.

Protein Extraction

For total protein isolation, leaves of B73 or mutant plants werecollected at the indicated times, quick-frozen in liquid nitrogen andground to a fine powder. One ml of extraction buffer (20 mM HEPES (pH7.6), 100 mM KCl, 10% Glycerol) was added to approximately 0.1 g frozenpowder and mixed thoroughly. Samples were centrifuged 10 min at 10,000rpm, the supernatant removed to a new tube and the concentrationdetermined spectrophotometrically according to the methods of Bradford(1976). See, Bradford M M (1976) A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principleof protein-dye binding. Anal. Biochem. 72:248-254. (See FIG. 18).

Chlorophyll Extraction

Leaves were frozen in liquid nitrogen and ground to a fine powder.Samples of approximately 0.1 g were removed to a 1.5 ml tube andweighed. Chlorophyll was extracted 5× with 1 ml (or 0.8 ml) of 80%acetone. Individual extractions were combined and the final volumeadjusted to 10 ml (or 15 ml) with additional 80% acetone. Chlorophyllcontent (a+b) was determined spectrophotometrically according to themethods of Wellburn (1994). See, Wellburn, A. R. (1994) The spectraldetermination of chlorophylls a and b, as well as total caretenoids,using various solvents with spectrophotometers of different resolution.J. Plant Physiol. 144:307-313. (See FIG. 17).

Measurement of Photosynthesis

Plants were grown in the field under normal and drought-stressconditions. Normal plants were watered for eight hours twice a week. Fordrought-stressed plants, water was limited to approximately four hoursper week for a period starting approximately one week before pollinationand continuing through three weeks after pollination. During the periodof limited water availability, drought-stressed plants showed visiblesigns of wilting and leaf rolling. Transpiration, stomatal conductanceand CO₂ assimilation were determined with a portable TPS-1Photosynthesis System (PP Systems). Each leaf on a plant was measured atforty days after pollination. Values represent a mean of sixdeterminations. See FIGS. 5 and 6.

DNA and RNA Purification

For total nucleic acid isolation, leaves of B73 are collected at desiredtimes, quick-frozen in liquid nitrogen and ground to a fine powder. Tenml of extraction buffer (100 mM Tris (pH 8.0), 50 mM EDTA, 200 mM NaCl,1% SDS, 10 μ/ml β-mercaptoethanol) is added and mixed thoroughly untilthawed. Ten ml of Phenol/Chloroform (1:1, vol:vol) is added and mixedthoroughly. Samples are centrifuged 10 min at 8,000 rpm, the supernatantis removed to a new tube and the nucleic acid is precipitated at −20° C.following addition of 1/10 vol 3M sodium acetate and 1 vol isopropanol.Total nucleic acid is pelleted by centrifugation at 8,000 rpm andresuspended in 1 ml TE. One half of the prep is used for DNApurification and the remaining half is used for RNA purification.(Alternatively, DNA or total nucleic acids can be extracted from 1 cm²of seedling leaf, quick-frozen in liquid nitrogen, and ground to a finepowder. 600 μl of extraction buffer [100 mM Tris (pH 8.0), 50 mM EDTA,200 mM NaCl, 1% SDS, 10 μl/ml β-mercaptoethanol] is added and the samplemixed. The sample is extracted with 700 μl phenol/chloroform (1:1) andcentrifuged for 10 min at 12,000 rpm. DNA is precipitated andresuspended in 600 μl H2O.)

For DNA purification, 500 μg DNase-free RNase is added to the tube andincubated at 37° C. for 1 hr. Following RNase digestion, an equal volumeof Phenol/Chloroform (1:1, vol:vol) is added and mixed thoroughly.Samples are centrifuged 10 min at 10,000 rpm, the supernatant is removedto a new tube and the DNA precipitated at −20° C. following addition of1/10 vol 3M sodium acetate and 1 vol isopropanol. DNA is resuspended insterile water and the concentration is determinedspectrophotometrically. To determine DNA integrity, 20 mg of DNA isseparated on a 1.8% agarose gel and visualized following staining withethidium bromide. RNA is purified by 2 rounds of LiCl₂ precipitationaccording to methods described by Sambrook et al, supra.

Real-Time RT-PCR Analysis

Fifty μg total RNA is treated with RQ1 DNase (Promega) to ensure that nocontaminating DNA is present. Two μg total RNA is used directly for cDNAsynthesis using the Omniscript RT kit (Qiagen) with oligo-dT(20) as theprimer.

Analysis of transcript abundance is accomplished using the QuantiTectSYBR Green PCR kit (Qiagen). Reactions contain 1× buffer, 0.5 μl of thereverse transcription reaction (equivalent to 50 ng total RNA) and 0.25μM (final concentration) forward and reverse primers (see table 2 below)in a total reaction volume of 25 μl. TABLE 2 Gene Forward Primer (5′-3′)Reverse Primer (5′-3′) ZmACS47 atcgcgtacagcctctccaaggagatagtcttttgtcaaccatcccataga SEQ ID NO: 19 SEQ ID NO: 20 ZmACS50atcgcgtacagcctctccaagga caacgtctctgtcactctgtgtaatgt SEQ ID NO: 21 SEQ IDNO: 22 ZmACS65 agctgtggaagaaggtggtcttcgaggt agtacgtgaccgtggtttctatga SEQID NO: 23 SEQ ID NO: 24

Reactions are carried out using an ABI PRISM 7700 sequence detectionsystem under the following conditions: 95° C./15 min. (1 cycle); 95°C./30 sec, 62° C./30 sec, 72° C./2 min (50 cycles); 72° C./5 min (1cycle). Each gene is analyzed a minimum of four times.

All the primer combinations are initially run and visualized on anagarose gel to confirm the presence single product of the correct size.All amplification products are subcloned into the pGEM-T Easy vectorsystem (Promega) to use for generation of standard curves to facilitateconversion of expression data to a copy/μg RNA basis.

Ethylene Determination

Ethylene was measured from the second fully-expanded leaf of seedlingsleaves at the 4-leaf stage or from the terminal 15 cm of leaves ofplants 20, 30, or 40 days after pollination (DAP). Leaves were harvestedat the indicated times and allowed to recover for 2 hr prior tocollecting ethylene, between moist paper towels. Leaves were placed intoglass vials and capped with a rubber septum. Following a 3-4 hourincubation, 0.9 mL of headspace was sampled from each vial and theethylene content measured using a 6850 series gas chromatography system(Hewlett-Packard, Palo Alto, Calif.) equipped with a HP Plotalumina-based capillary column (Agilent Technologies, Palo Alto,Calif.). Tissue fresh weight was measured for each sample. Threereplicates were measured and the average and standard deviationreported.

Western Blot Analysis

B73 leaves were collected at the indicated times and ground in liquidnitrogen to a fine powder. One ml of extraction buffer [20 mM HEPES (pH7.6), 100 mM KCl, 10% glycerol, 1 mM PMSF] was added to approximately0.1 g frozen powder and mixed thoroughly. Cell debris was pelleted bycentrifugation at 10,000 rpm for 10 min and the protein concentrationdetermined as described (Bradford, 1976). Antiserum raised against thelarge subunit of rice Rubisco was obtained from Dr. Tadahiko Mae (TohokuUniversity, Sendai, Japan). Protein extracts were resolved usingstandard SDS-PAGE and the protein transferred to 0.22 μm nitrocellulosemembrane by electroblotting. Following transfer, the membranes wereblocked in 5% milk, 0.01% thimerosal in TPBS (0.1% TWEEN 20, 13.7 mMNaCl, 0.27 mM KCl, 1 mM Na2HPO4, 0.14 mM KH2PO4) followed by incubationwith primary antibodies diluted typically 1:1000 to 1:2000 in TPBS with1% milk for 1.5 hrs. The blots were then washed twice with TPBS andincubated with goat anti-rabbit horseradish peroxidase-conjugatedantibodies (Southern Biotechnology Associates, Inc.) diluted to 1:5000to 1:10,000 for 1 hr. The blots were washed twice with TPBS and thesignal detected typically between 1 to 15 min using chemiluminescence(Amersham Corp).

Results

Identification of ACC Synthase Knockout Mutants

Three genes encoding ACC synthase were isolated from the inbred B73 andsequenced (see, e.g., SEQ ID NOs:1-11). Two members of the family (i.e.,ACS2 and ACS7) are closely related (97% amino acid identity) whereas thethird gene (i.e., ACS6) is considerably more divergent (54% and 53%amino acid identity with ACS2 and ACS7, respectively). A reverse geneticapproach was used to screen for transposon insertions in ACC synthasegene family members (Bensen et al. (1995) Cloning and characterizationof the maize An1 gene. Plant Cell 7:75-84). 19 candidate lines wereidentified, 13 of which were confirmed by terminal-inverted-repeat(TIR)-PCR to harbor a Mu insertion in one of the three ACC synthasegenes. Of these, 5 lines stably inherited the transposon in the firstbackcross to B73 which were backcrossed an additional 4 times to reduceunwanted Mu insertions. Plants were then self-pollinated to generatehomozygous null individuals which were identified by PCR using the ACCF1and ACC-1 primers (see Methods). PCR amplification of wild-type lines orheterozygous null mutants with these primers resulted in three differentsized fragments corresponding to the three ACC synthase genes whereasthe products of PCR amplification of homozygous null mutants lack thefragment corresponding to the mutant gene. The Mu insertion site foreach mutant line was determined by sequencing across the Mu/ACC synthasejunction using the Mu-TIR primer (FIG. 2). Four of the five insertionlines contained a Mu in ACS2: one mutant contained an insertion in thethird exon whereas the other three contained insertions in the fourthexon at unique positions (FIG. 2). The fifth insertion line contained aMu in ACS6 in the second intron near the 3′ splice site. Quantitativereal time RT-PCR revealed that all three genes are expressed duringmaize leaf development and confirmed that the Mu insertions resulted inthe loss of or a decrease in ACS expression. Insertions in ACS7 wereidentified in the first generation but were not inherited, suggestingthat they were somatic mutants or that expression of ACS7 is requiredfor germ line development.

For a description of ACC synthase expression patterns during endospermand embryo development, see Gallie and Young (2004) The ethylenebiosynthetic and perception machinery is differentially expressed duringendosperm and embryo development Mol Gen Genomics 271:267-281.

ACS6 or ACS2 Gene Disruption Reduces Ethylene Synthesis

The level of ethylene evolution in maize leaves increased as a functionof leaf age (FIG. 19 Panel C). At 20 DAP, the highest level of ethylenewas observed in leaf 1 (the oldest surviving leaf) which by 30 DAP hadprogressed to leaf 3 and by 40 DAP (i.e., kernel maturity) to leaves4-5. To determine whether Mu disruptions of ACS6 or ACS2 described abovereduced ethylene evolution, ethylene was measured from leaf 4 ofwild-type and mutant plants. Ethylene evolution from acs2 plants wasapproximately 55% of wild-type plants, a level that was similar for allacs2 mutant alleles (FIG. 19 Panels A-B). Ethylene evolution from acs6plants was only 10% of that from wild-type plants (FIG. 19 Panel B).Ethylene evolution from acs2/acs6 double mutant plants was similar tothat from acs6 plants. These data suggest that loss of ACS6 expressionresults in a greater reduction in the ability of maize leaves to produceethylene than does the loss of ACS2 expression.

Disruption of ACS6 Confers a Staygreen Phenotype

A substantial increase in ethylene evolution correlated with theappearance of visible signs of senescence in wild-type leaves,suggesting that ethylene may promote the entry of leaves into thesenescence program. If so, a delay in the senescence of acs6 leaves,which produce significantly less ethylene, would be expected. To testthis possibility, homozygous (i.e., acs6/acs6), heterozygous (i.e.,ACS6/acs6), and wild-type (i.e., ACS6/ACS6) plants were field-grownuntil 50 days after pollination. At this stage, the oldest wild-typeleaves had senesced, whereas the corresponding ACS6/acs6 leaves werejust beginning to senesce and acs6/acs6 leaves remained fully green.These observations suggest that the level of ethylene evolution maydetermine the timing of leaf senescence.

Senescence can also be induced following prolonged exposure to darkness.To determine whether a reduction in ethylene evolution can delaydark-induced senescence, leaves from adult plants were covered withsheaths to exclude light for two weeks. The leaves from younger plants(i.e., 20 DAP) were used to ensure that age-related senescence would notoccur during the course of the experiment and they remained attached tothe plant. Greenhouse grown maize was also employed to avoid any heatingthat might occur in the field as a consequence of the sheathing.Following the two-week dark-treatment, senescence was observed forvirtually the entire region of wild-type leaves that was covered (theregion covered by the sheath is indicated by the distinct transitionfrom yellow to green, FIG. 4 on left). The tip of ACS6/acs6 leaves hadundergone dark-induced senescence but the rest of the covered regionshowed significantly less senescence (FIG. 4 in center). In contrast,acs6/acs6 leaves remained fully green (FIG. 4 on right). The degree ofsenescence correlated with the amount of ethylene produced by each, inwhich ACS6/acs6 leaves produced just 70% of wild-type ethylene andacs6/acs6 leaves produced only 14.6% of wild-type ethylene. Theseresults suggest that ethylene mediates the onset of dark-inducedsenescence as it does natural senescence. They also indicate that theACS6/acs6 heterozygous mutant with a loss of one copy of ACS6 producesless ethylene and exhibits a weak staygreen phenotype similar to thatobserved for the acs2 mutant which also exhibited a moderate (i.e., 40%)reduction in ethylene evolution.

To examine if exogenous ACC could complement the acs6 mutant and reverseits staygreen phenotype, the third oldest, sixth, and ninth leaf fromACS6/ACS6, acs2/acs2, and acs6/acs6 plants were subject to dark-inducedsenescence at 20 DAP by covering them with sheaths for 7 days. Allleaves were fully green at the onset of the experiment and remainedattached to the plant. acs6/acs6 plants were watered daily with water or100 μM ACC for 7 days. Following 7 days, dark-induced senescence hadinitiated in wild-type (i.e., ASC6/ASC6) leaves although it had notprogressed to the extent observed following a 2 week dark treatment. Theextent of dark-induced senescence increased as a function of leaf agesuch that leaf 3 exhibited more senescence than did leaf 6 or leaf 9(which were younger), suggesting that competency for senescenceincreases with leaf age. Dark-induced senescence was also observed inleaf 3 of the acs2 homozygous mutant although it was less pronouncedthan that observed in the corresponding wild-type leaves. Although noacs6 homozygous leaves exhibited dark-induced senescence consistent withthe observations made in FIG. 4, dark-induced senescence similar to thatof wild-type leaves was observed when acs6 leaves were watered with 100μM ACC for 7 days. The ACC treatment did not affect acs6 leaves thatwere not sheathed, demonstrating that the senescence observed forsheathed acs6 leaves was specific to the dark-treatment.

Determination of the level of chlorophyll a+b from leaf 3 confirmed thevisual results in that acs6 leaves retained substantially morechlorophyll after the 7 day dark treatment than did wild-type leaves butdid not do so when watered with 100 μM ACC (FIG. 20 Panel A). Thetreatment with ACC in itself did not induce premature loss ofchlorophyll as chlorophyll was not lost from unsheathed leaves of acs6mutants watered with ACC. acs2 leaves retained only moderately greateramount of chlorophyll did wildtype leaves. Similar results were observedfor leaf 6 and leaf 9 although the level of chlorophyll in these youngerleaves was higher than in the older leaf 3 samples as was expected (FIG.20 Panel A). Similar trends were observed for total soluble leafprotein: acs6 leaves retained substantially more protein following thedark treatment than did wild-type leaves but did not do so when wateredwith 100 μM ACC (FIG. 20 Panel B).

Western analysis for ribulose biscarboxylase (Rubisco) demonstrated asubstantial loss of Rubisco from dark-treated B73 leaves that wasgreater with the oldest leaves (leaf 3) than the youngest (leaf 9) (FIG.20 Panel C). Dark-treated acs6 leaves retained substantially moreRubisco than did dark-treated wild-type leaves and acs2 leaves retaineda moderate level of Rubisco (FIG. 20 Panel C). Dark-treated acs6 leaveswatered with 100 μM ACC lost an amount of Rubisco similar to that ofdark-treated B73 leaves suggesting that ACC complemented the loss of ACCsynthase expression. No loss of Rubisco was observed in ACC-treated acs6leaves when they remained in the light demonstrating that treatment withACC alone did not reduce the level of Rubisco. These data demonstratethat the staygreen phenotype, which involves retention of chlorophylland leaf protein such as Rubisco, can be complemented by exogenous ACC,suggesting that the delay in senescence in these plants is a consequenceof the reduction in loss of ACC synthase expression in the acs6 mutant.

Reducing Ethylene Delays Natural Leaf Senescence and Reduces Loss ofChlorophyll and Protein

Drought is known to induce premature onset of leaf senescence. Toinvestigate whether the drought response is mediated by ethylene and todetermine whether reducing ethylene evolution may increase droughttolerance in maize, homozygous acs6 and acs2 mutant plants and wild-typeplants were field-grown under well-watered (eight hours twice a week)and water-stressed conditions (four hours per week for a one monthperiod that initiated approximately one week before pollination andcontinued for 3 weeks after pollination). During the period of limitedwater availability, drought-stressed plants exhibited leaf wilting androlling, visible confirmation of drought stress. Following the waterstress treatment, the extent of leaf senescence and function wasmeasured.

Senescence of the oldest leaves was evident in wild-type plants underwell-watered conditions and even more significantly during droughtconditions. Similar results were observed for acs2 plants. In contrast,no visible sign of senescence was observed in acs6 leaves underwell-watered or drought conditions. Interestingly, anthocyaninproduction was also reduced in acs6 leaves.

To confirm that the staygreen phenotype correlates with enhanced levelsof chlorophyll, the level of chlorophyll a and b was measured.Chlorophyll decreased with leaf age as well as with the age of theplants (FIG. 17 Panels A and D). As expected, the greatest decrease inchlorophyll correlated with the visible onset of senescence. Underwell-watered conditions, the level of chlorophyll in acs6 (ACS6 0/0)leaves was up to 8-fold higher than in the corresponding leaves ofwild-type plants that had initiated senescence. Surprisingly, the levelof chlorophyll in all acs6 leaves, including the youngest, wassubstantially higher than in wild-type plants (FIG. 17 Panel A). Thelevel of chlorophyll in acs2 (ACS2 0/0) leaves was moderately higherthan in wild-type plants. These results indicate that the increase inchlorophyll content inversely correlates with the level of ethyleneproduction: the moderate reduction in ethylene in acs2 plants correlatedwith a moderate increase in chlorophyll content whereas the largereduction in ethylene in acs6 plants correlated with a substantialincrease in chlorophyll content. These results also demonstrate thatreducing ethylene increases the level of chlorophyll even in youngleaves that are exhibiting maximum leaf function (see below).

Under drought conditions, the level of chlorophyll was reduced in mutantand wild-type plants but decreased to an even greater extent inwild-type plants (FIG. 17 Panels B-C). For example, the level ofchlorophyll in leaf 5 of water-stressed wild-type plants decreased2.5-fold relative to non-drought plants whereas it decreased by only 20%in leaf 5 of water-stressed acs6 plants (FIG. 17 Panel C). Consequently,reducing ethylene evolution resulted in a level of chlorophyll in theoldest leaves of acs6 plants that was up to 20-fold higher than in thecorresponding leaves of wild-type plants. As observed for non-droughtplants, the level of chlorophyll was higher in all acs6 leaves,including the youngest. Chlorophyll content in acs2 leaves also remainedmoderately higher under drought conditions than in wild-type plants.Thus, loss of ACS6 expression reduced responsiveness to water stress inthat chlorophyll content was substantially maintained under those stressconditions that had elicited a significant loss of chlorophyll inwild-type plants.

Leaf protein also declined with leaf age and with plant age (FIG. 18Panel D). As observed for chlorophyll, the most substantial decrease inprotein correlated with the visible onset of senescence (FIG. 18 PanelD). Under non-drought conditions, the level of protein in acs6 leaveswas up to 2-fold higher than in the corresponding leaves of wild-typeplants that had initiated senescence (FIG. 18 Panel A). As observed forchlorophyll, the level of protein in all acs6 leaves, including theyoungest, was substantially higher than in wild-type plants and thelevel of protein in acs2 leaves was moderately higher than in wild-typeplants (FIG. 18 Panel A). Exposure to conditions of drought resulted ina greater decrease of protein in the oldest wildtype leaves than wasobserved in acs6 leaves (FIG. 18 Panels B-C). As observed fornon-drought plants, the level of protein was higher in all acs6 leaves,including the youngest. These results parallel those for chlorophyll andindicate that protein content inversely correlates with the level ofethylene evolution. They also demonstrate that loss of ACC synthaseexpression in the acs6 mutant reduced responsiveness to water stress inthat protein levels were substantially maintained under those stressconditions that had elicited a significant reduction of protein inwild-type plants.

Reducing Ethylene Maintains Leaf Function During Well-Watered andDrought Conditions

The maintenance of chlorophyll and protein in acs6 leaves suggests thatleaf function, e.g., the ability to transpire and assimilate CO₂, mayalso be maintained. To investigate this, the rate of transpiration,stomatal conductance, and rate of CO₂ assimilation were measured inevery leaf of well watered acs6 and wild-type plants at 40 DAP when thelower leaves of wild-type plants had begun to senesce. The youngestleaves of acs6 plants exhibited a higher rate of transpiration (FIG. 5Panel A) and stomatal conductance (FIG. 5 Panel B) than control plantswhereas no significant difference was observed in older leaves. Incontrast, the rate of CO₂ assimilation was substantially higher in allleaves of acs6 plants than in control plants (FIG. 5 Panel C).Specifically, older leaves of acs6 plants exhibited more than a 2-foldhigher rate of CO₂ assimilation than wild-type plants and the rate ofCO₂ assimilation in younger leaves increased from 50 to 100% (FIG. 5Panel C).

The effect of reducing ethylene on the maintenance of leaf functionunder drought conditions was also investigated. The rate oftranspiration (FIG. 5 Panel D) and stomatal conductance (FIG. 5 Panel E)were significantly reduced in wild-type leaves when subjected toconditions of drought (i.e., four hours per week for a one month periodstarting approximately one week before pollination and continuingthrough three weeks after pollination) whereas they remained largelyunaffected in acs6 leaves, resulting in a substantially higher rate oftranspiration (FIG. 5 Panel D) and increased stomatal conductance (FIG.5 Panel E) for the mutant. In addition, drought treatment resulted in asignificant decrease in the rate of CO₂ assimilation in wild-type leavesbut not in acs6 leaves, resulting in up to a 2.5-fold increase in CO₂assimilation in younger acs6 leaves and up to a 6-fold increase in olderacs6 leaves than in the control (FIG. 5 Panel F). These results indicatethat ethylene controls leaf function during conditions of drought and areduction in its production results in a delay of leaf senescence inolder leaves while maintaining leaf function in all leaves thusproviding greater tolerance to drought. Similar, though less pronounced,results were obtained for ACS2 (FIG. 6 Panels A-C).

Discussion

In summary, ACC synthase mutants affecting the first step in ethylenebiosynthesis were isolated in maize. These mutants exhibited a delay innatural, dark-induced, and drought-induced leaf senescence and astaygreen phenotype. The delay in senescence was reversible followingexposure to ethylene. ACC synthase mutant leaves exhibited asubstantially higher rate of CO₂ assimilation during growth under normalor drought conditions. Surprisingly, improved leaf function was observedin all ACC synthase mutant leaves, including the youngest which had notentered either natural or drought-induced senescence programs. Theseobservations suggest that ethylene mediates the response of maize towater stress and that decreasing ethylene production serves as a meansto maintain leaf performance during water stress and thereby increaseits tolerance to drought conditions. As noted, ACC synthase mutants canhave other advantageous phenotypes, e.g., male sterility phenotypes,crowding resistance phenotypes, altered pathogen resistance, and thelike.

The above examples show that ethylene plays a significant role inregulating the onset of leaf senescence in maize whether during growthunder well-watered conditions or during conditions of drought whichnormally induces premature leaf senescence. The reduction in ethyleneevolution resulting from loss of ACS6 expression is largely responsiblefor directing natural and drought-induced leaf senescence. While notintending to be limited by any particular theory, loss of ACS6expression may directly delay entry into the senescence program or mayaffect total ACC synthase expression from all gene members. Knockout ofACS2 alone reduced ethylene production by approximately 40% and didresult in a small increase in chlorophyll and protein. In contrast,ethylene production in acs6 leaves was reduced up to 90% and acs6 leavescontained substantially higher levels of chlorophyll and protein. Theseobservations suggests that entry into the senescence program may becontrolled by more than one gene family member.

The level of chlorophyll and protein in wild-type leaves was reducedsubstantially following water-stress but remained unaffected in acs6leaves. These results indicate two roles for ethylene in maize leaves:under normal growth conditions, ethylene may help to maintain thecorrect level of chlorophyll and protein in a leaf, whereas during waterstress, ethylene may serve to reduce the level of both. The observationthat a 40% reduction in ethylene resulted in a moderate increase inchlorophyll and protein whereas a 90% reduction resulted in asubstantially larger increase in chlorophyll and protein suggests thatthese leaf components may be quantitatively controlled by the level ofethylene produced in leaves. Greater increases in leaf chlorophyll andprotein might be expected if ethylene production were reduced evenfurther.

Loss of chlorophyll and protein in wild-type maize subjected to droughtconditions was accompanied by decreased rates of transpiration, stomatalconductance, and CO₂ assimilation. In contrast, maintenance ofchlorophyll and protein levels in leaves of acs6 plants subjected todrought conditions was accompanied by the maintenance of transpiration,stomatal conductance, and CO₂ assimilation. These results suggest thatreducing ethylene not only confers a staygreen phenotype but actuallymaintains leaf function under stress conditions. The observation thatethylene controls the onset of leaf senescence is consistent with therole of this hormone in other species such as Arabidopsis and tomato(Davis and Grierson (1989) Identification of cDNA clones for tomato(Lycopersicon esculentum Mill.) mRNAs that accumulate during fruitripening and leaf senescence in response to ethylene. Planta 179:73-80;Abeles et al. (1992). Ethylene in Plant Biology. (San Diego:AcademicPress); Picton et al. (1993). Altered fruit ripening and leaf senescencein tomatoes expressing an antisense ethylene-forming enzyme transgenePlant J. 3:469-481; Grbic and Bleecker (1995) Ethylene regulates thetiming of leaf senescence in Arabidopsis Plant J. 8:95-102; John et al.(1995) Delayed leaf senescence in ethylene-deficient ACC-oxidaseantisense tomato plants: molecular and physiological analysis Plant J.7:483-490). The observation that a reduction in ethylene evolution wouldincrease the level of chlorophyll and protein and increase the rate ofCO₂ assimilation in all leaves, including the youngest, was unexpected.This suggests that ethylene plays an active role in controlling aspectsof leaf function well before a leaf enters a senescence program. Equallyunexpected was the observation that a reduction in ethylene would affectthe water-stress response of all leaves. These findings suggest thatincreased tolerance to conditions of drought can be easily introducedinto maize, and optionally other grain species, through a reduction inthe level of ethylene produced in leaves.

Example 2 Sequence Alignments and Phylogenetic Analysis

A phylogenetic analysis of ACC synthase sequences described herein,e.g., (A47 (also known as ACS2 or ACC2 herein), A50 (also known as ACS7or ACC7 herein), A65 (also known as ACS6 or ACC6 herein)) from maize,with ACC synthase sequences from other species, is shown in FIG. 7(ACSgrowtree2), where Arabidopsis sequences are indicated by (AtACS . .. ), tomato sequences are indicated by (LeACS . . . ), rice sequencesare indicated by (indica (OsiACS . . . ), and japonica (OsjACS . . . )),wheat sequences are indicated by (TaACS . . . ), and banana sequencesare indicated by (MaACS . . . ). In the analysis, the indicated ACCsynthases fall into two subfamilies. One of the subfamilies is furthersubdivided into monocot (Zm (maize), Osi, Osj, Ta, Ma) ACS genes anddicot (At, Le) ACS genes.

Various peptide consensus sequences alignments of ACC synthase sequencesdescribed herein, e.g., (A47 (also known as ACS2 or ACC2 herein), A50(also known as ACS7 or ACC7 herein), A65 (also known as ACS6 or ACC6herein)) from maize (Zm), with ACC synthase sequences from other speciesare shown in FIGS. 8-16. A Pretty program is used (e.g., available onthe SeqWeb (GCG) web page) to determine the consensus sequence withdifferent stringencies (e.g., most stringent (identical), stringent(similar amino acids), or least stringent (somewhat similar aminoacids). The stringency is indicated in each figure after “consensussequence.” The GapWeight is 8 and the GapLengthWeight is 2.

Example 3 ACC Synthase Knockouts by Hairpin RNA Expression

As noted previously, knockout plant cells and plants can be produced,for example, by introduction of an ACC synthase polynucleotide sequenceconfigured for RNA silencing or interference. This example describeshairpin RNA expression cassettes for modifying ethylene production andstaygreen phenotype, e.g., in maize. As noted previously, knockout ofACC synthase(s), e.g., by hpRNA expression, can result in plants orplant cells having reduced expression (up to and including no detectableexpression) of one or more ACC synthases.

Expression of hairpin RNA (hpRNA) molecules specific for ACC synthasegenes (e.g., promoters, other untranslated regions, or coding regions)that encode ACC synthases in plants can alter ethylene production andstaygreen potential, sterility, crowding resistance, etc. of the plants,e.g., through RNA interference and/or silencing.

hpRNA constructs of ACS2 (PHP20600) and ACS6 (PHP20323) were generatedby linking a ubiquitin promoter to an inverted repeat of a portion ofthe coding sequence of either the ACS2 or ACS6 gene (see FIGS. 21 and22, Panels A-C). Each construct was transformed into maize usingAgrobacterium-mediated transformation techniques. Nucleic acid moleculesand methods for preparing the constructs and transforming maize were aspreviously described and known in the art; see, e.g., the sectionsherein entitled “Vectors, Promoters, and Expression Systems,” “PlantTransformation,” “Other Nucleic Acid and Protein Assays,” and thefollowing example “Transformation of Maize”.

Expression of hpRNA specific for either ACS2 or ACS6 coding sequencesresulted in maize plants that displayed no abnormalities in vegetativeand reproductive growth. A total of 36 and 40 individual maizetransgenic events were generated for ACS2- and ACS6-hairpin constructs,respectively (FIG. 23, Panels A and B).

Approximately 10 low copy number events per hpRNA construct wereselected for additional backcrossing and transgene evaluation. Staygreenpotential phenotype is evaluated for the backcrossed lines comprisingthe hpRNA transgene(s), e.g., as described herein (for example, byvisual inspection, measurements of photosynthetic activity,determination of chlorophyll or protein content, or the like, undernormal and drought or other stress conditions).

Example 4 Transformation of Maize

Biolistics

The inventive polynucleotides contained within a vector are transformedinto embryogenic maize callus by particle bombardment, generally asdescribed by Tomes, D. et al., IN: Plant Cell, Tissue and Organ Culture:Fundamental Methods, Eds. O. L. Gamborg and G. C. Phillips, Chapter 8,pgs. 197-213 (1995) and as briefly outlined below. Transgenic maizeplants are produced by bombardment of embryogenically responsiveimmature embryos with tungsten particles associated with DNA plasmids.The plasmids typically comprise or consist of a selectable marker and anunselected structural gene, or a selectable marker and an ACC synthasepolynucleotide sequence or subsequence, or the like.

Preparation of Particles:

Fifteen mg of tungsten particles (General Electric), 0.5 to 1.8μ,preferably 1 to 1.8μ, and most preferably 1μ, are added to 2 ml ofconcentrated nitric acid. This suspension is sonicated at 0° C. for 20minutes (Branson Sonifier Model 450, 40% output, constant duty cycle).Tungsten particles are pelleted by centrifugation at 10000 rpm (Biofuge)for one minute, and the supernatant is removed. Two milliliters ofsterile distilled water are added to the pellet, and brief sonication isused to resuspend the particles. The suspension is pelleted, onemilliliter of absolute ethanol is added to the pellet, and briefsonication is used to resuspend the particles. Rinsing, pelleting, andresuspending of the particles is performed two more times with steriledistilled water, and finally the particles are resuspended in twomilliliters of sterile distilled water. The particles are subdividedinto 250-μl aliquots and stored frozen.

Preparation of Particle-Plasmid DNA Association:

The stock of tungsten particles are sonicated briefly in a water bathsonicator (Branson Sonifier Model 450, 20% output, constant duty cycle)and 50 μl is transferred to a microfuge tube. The vectors are typicallycis: that is, the selectable marker and the gene (or otherpolynucleotide sequence) of interest are on the same plasmid.

Plasmid DNA is added to the particles for a final DNA amount of 0.1 to10 μg in 10 μL total volume, and briefly sonicated. Preferably, 10 μg (1μg/μL in TE buffer) total DNA is used to mix DNA and particles forbombardment. Fifty microliters (50 μL) of sterile aqueous 2.5 M CaCl₂are added, and the mixture is briefly sonicated and vortexed. Twentymicroliters (20 μL) of sterile aqueous 0.1 M spermidine are added andthe mixture is briefly sonicated and vortexed. The mixture is incubatedat room temperature for 20 minutes with intermittent brief sonication.The particle suspension is centrifuged, and the supernatant is removed.Two hundred fifty microliters (250 μL) of absolute ethanol are added tothe pellet, followed by brief sonication. The suspension is pelleted,the supernatant is removed, and 60 μl of absolute ethanol are added. Thesuspension is sonicated briefly before loading the particle-DNAagglomeration onto macrocarriers.

Preparation of Tissue

Immature embryos of maize variety High Type II are the target forparticle bombardment-mediated transformation. This genotype is the F₁ oftwo purebred genetic lines, parents A and B, derived from the cross oftwo known maize inbreds, A188 and B73. Both parents are selected forhigh competence of somatic embryogenesis, according to Armstrong et al.,Maize Genetics Coop. News 65:92 (1991).

Ears from F₁ plants are selfed or sibbed, and embryos are asepticallydissected from developing caryopses when the scutellum first becomesopaque. This stage occurs about 9-13 days post-pollination, and mostgenerally about 10 days post-pollination, depending on growthconditions. The embryos are about 0.75 to 1.5 millimeters long. Ears aresurface sterilized with 20-50% Clorox for 30 minutes, followed by threerinses with sterile distilled water.

Immature embryos are cultured with the scutellum oriented upward, onembryogenic induction medium comprised of N6 basal salts, Erikssonvitamins, 0.5 mg/l thiamine HCl, 30 gm/l sucrose, 2.88 gm/l L-proline, 1mg/l 2,4-dichlorophenoxyacetic acid, 2 gm/l Gelrite, and 8.5 mg/l AgNO₃.Chu et al., Sci. Sin. 18:659 (1975); Eriksson, Physiol. Plant 18:976(1965). The medium is sterilized by autoclaving at 121° C. for 15minutes and dispensed into 100×25 mm Petri dishes. AgNO₃ isfilter-sterilized and added to the medium after autoclaving. The tissuesare cultured in complete darkness at 28° C. After about 3 to 7 days,most usually about 4 days, the scutellum of the embryo swells to aboutdouble its original size and the protuberances at the coleorhizalsurface of the scutellum indicate the inception of embryogenic tissue.Up to 100% of the embryos display this response, but most commonly, theembryogenic response frequency is about 80%.

When the embryogenic response is observed, the embryos are transferredto a medium comprised of induction medium modified to contain 120 gm/lsucrose. The embryos are oriented with the coleorhizal pole, theembryogenically responsive tissue, upwards from the culture medium. Tenembryos per Petri dish are located in the center of a Petri dish in anarea about 2 cm in diameter. The embryos are maintained on this mediumfor 3-16 hour, preferably 4 hours, in complete darkness at 28° C. justprior to bombardment with particles associated with plasmid DNAscontaining the selectable and unselectable marker genes.

To effect particle bombardment of embryos, the particle-DNA agglomeratesare accelerated using a DuPont PDS-1000 particle acceleration device.The particle-DNA agglomeration is briefly sonicated and 10 μl aredeposited on macrocarriers and the ethanol is allowed to evaporate. Themacrocarrier is accelerated onto a stainless-steel stopping screen bythe rupture of a polymer diaphragm (rupture disk). Rupture is effectedby pressurized helium. The velocity of particle-DNA acceleration isdetermined based on the rupture disk breaking pressure. Rupture diskpressures of 200 to 1800 psi are used, with 650 to 1100 psi beingpreferred, and about 900 psi being most highly preferred. Multiple disksare used to effect a range of rupture pressures.

The shelf containing the plate with embryos is placed 5.1 cm below thebottom of the macrocarrier platform (shelf #3). To effect particlebombardment of cultured immature embryos, a rupture disk and amacrocarrier with dried particle-DNA agglomerates are installed in thedevice. The He pressure delivered to the device is adjusted to 200 psiabove the rupture disk breaking pressure. A Petri dish with the targetembryos is placed into the vacuum chamber and located in the projectedpath of accelerated particles. A vacuum is created in the chamber,preferably about 28 in Hg. After operation of the device, the vacuum isreleased and the Petri dish is removed.

Bombarded embryos remain on the osmotically-adjusted medium duringbombardment, and 1 to 4 days subsequently. The embryos are transferredto selection medium comprised of N6 basal salts, Eriksson vitamins, 0.5mg/l thiamine HCl, 30 gm/l sucrose, 1 mg/l 2,4-dichlorophenoxyaceticacid, 2 gm/l Gelrite, 0.85 mg/l Ag NO₃ and 3 mg/l bialaphos (Herbiace,Meiji). Bialaphos is added filter-sterilized. The embryos aresubcultured to fresh selection medium at 10 to 14 day intervals. Afterabout 7 weeks, embryogenic tissue, putatively transformed for bothselectable and unselected marker genes, proliferates from about 7% ofthe bombarded embryos. Putative transgenic tissue is rescued, and thattissue derived from individual embryos is considered to be an event andis propagated independently on selection medium. Two cycles of clonalpropagation are achieved by visual selection for the smallest contiguousfragments of organized embryogenic tissue.

A sample of tissue from each event is processed to recover DNA. The DNAis restricted with a restriction endonuclease and probed with primersequences designed to amplify DNA sequences overlapping the ACC synthaseand non-ACC synthase portion of the plasmid. Embryogenic tissue withamplifiable sequence is advanced to plant regeneration.

For regeneration of transgenic plants, embryogenic tissue is subculturedto a medium comprising MS salts and vitamins (Murashige & Skoog,Physiol. Plant 15:473 (1962)), 100 mg/l myo-inositol, 60 gm/l sucrose, 3gm/l Gelrite, 0.5 mg/l zeatin, 1 mg/l indole-3-acetic acid, 26.4 ng/lcis-trans-abscissic acid, and 3 mg/l bialaphos in 100×25 mm Petridishes, and is incubated in darkness at 28° C. until the development ofwell-formed, matured somatic embryos can be seen. This requires about 14days. Well-formed somatic embryos are opaque and cream-colored, and arecomprised of an identifiable scutellum and coleoptile. The embryos areindividually subcultured to a germination medium comprising MS salts andvitamins, 100 mg/l myo-inositol, 40 gm/l sucrose and 1.5 gm/l Gelrite in100×25 mm Petri dishes and incubated under a 16 hour light:8 hour darkphotoperiod and 40 meinsteinsm⁻²sec⁻¹ from cool-white fluorescent tubes.After about 7 days, the somatic embryos have germinated and produced awell-defined shoot and root. The individual plants are subcultured togermination medium in 125×25 mm glass tubes to allow further plantdevelopment. The plants are maintained under a 16 hour light:8 hour darkphotoperiod and 40 meinsteinsm⁻²sec−1 from cool-white fluorescent tubes.After about 7 days, the plants are well-established and are transplantedto horticultural soil, hardened off, and potted into commercialgreenhouse soil mixture and grown to sexual maturity in a greenhouse. Anelite inbred line is used as a male to pollinate regenerated transgenicplants.

Agrobacterium-Mediated

When Agrobacterium-mediated transformation is used, the method of Zhaois employed as in PCT patent publication WO98/32326, the contents ofwhich are hereby incorporated by reference. Briefly, immature embryosare isolated from maize and the embryos contacted with a suspension ofAgrobacterium (step 1: the infection step). In this step the immatureembryos are preferably immersed in an Agrobacterium suspension for theinitiation of inoculation. The embryos are co-cultured for a time withthe Agrobacterium (step 2: the co-cultivation step). Preferably theimmature embryos are cultured on solid medium following the infectionstep. Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step) and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants.

Example 5 Expression of Transgenes in Monocots

A plasmid vector is constructed comprising a preferred promoter operablylinked to an isolated polynucleotide comprising an ACC synthasepolynucleotide sequence or subsequence (e.g., selected from SEQ IDNOs:1-6 and 10). This construct can then be introduced into maize cellsby the following procedure.

Immature maize embryos are dissected from developing caryopses derivedfrom crosses of maize lines. The embryos are isolated 10 to 11 daysafter pollination when they are 1.0 to 1.5 mm long. The embryos are thenplaced with the axis-side facing down and in contact withagarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking18:659-668). The embryos are kept in the dark at 27° C. Friableembryogenic callus, consisting of undifferentiated masses of cells withsomatic proembryoids and embryoids borne on suspensor structures,proliferates from the scutellum of these immature embryos. Theembryogenic callus isolated from the primary explant can be cultured onN6 medium and sub-cultured on this medium every 2 to 3 weeks.

The plasmid p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent maybe used in transformation experiments in order to provide for aselectable marker. This plasmid contains the Pat gene (see EuropeanPatent Publication 0 242 236) which encodes phosphinothricin acetyltransferase (PAT). The enzyme PAT confers resistance to herbicidalglutamine synthetase inhibitors such as phosphinothricin. The pat genein p35S/Ac is under the control of the 35S promoter from CauliflowerMosaic Virus (Odell et al. (1985) Nature 313:810-812) and comprises the3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmidof Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated gold particles can be placed in thecenter of a Kapton flying disc (Bio-Rad Labs). The particles are thenaccelerated into the corn tissue with a Biolistic PDS-1000/He (Bio-RadInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovers a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 6 Expression of Transgenes in Dicots

Soybean embryos are bombarded with a plasmid comprising a preferredpromoter operably linked to a heterologous nucleotide sequencecomprising an ACC synthase polynucleotide sequence or subsequence (e.g.,selected from SEQ ID NOs:1-6 and 10), as follows. To induce somaticembryos, cotyledons of 3-5 mm in length are dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, thencultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiply as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescentlights on a 16:8 hour day/night schedule. Cultures are sub-culturedevery two weeks by inoculating approximately 35 mg of tissue into 35 mlof liquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic PDS1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette of interest, comprising thepreferred promoter and a heterologous ACC synthase polynucleotide, canbe isolated as a restriction fragment. This fragment can then beinserted into a unique restriction site of the vector carrying themarker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×5 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes. SEQ ID: TYPE SEQUENCE SEQ ID: 1 Gene ACS2(ACC2) AAACTTCATA CCGGTCGGTG CCTTACGTTC TCTGGCGTTC TTATCCTTTC ACCSynthase gene CTCCGCTTTT AGTCGATGAT TATAGTAGTT TCTACAACAA GCTTTCAACGsequence (A47) CCATTGACTA TTTTTTCCCC CATTGAAAAC GAACACCACC ATTGACACTGfrom Zea mays (Zm) ATAAATGTAG TACAGCATTT GACAACATAC TTTCCTAGAAAGTAACCAGC inbred ‘B73’ (− AGAGACTGGA CGCTACGTAC TACCACACCA TTGGAGCAGCCAATTTAATC ACC2 pcr fragment GTGTATAGAA CTCCGTATCG AAATTTGTCT GTGAATGGACCTTCATTTGC originally ATCTAGGTCT AGTACAATGG ATTTCGAACA GGACAGCGCCGATCTGGCAA isolated from TACACACACG CACGACGTAG CACAGCTGTT CTTCGTTCCACGCGTTAATT inbred ‘Oh43’) GAAGGCAAAG CGACTGTAGT TGCTGTTGGT GGCCAAGTTGTTTAATGCTA TAGTAGCAGC CAGTCACTCC TAGGGCAAAT TTTAGGACTT TTGCATTGCATTGCCGCCAT GTAGAGGTTG ACTGCACACC GAGAATATCG ACCATTCATT AGGCTCCTTGACTTGTTGCT GTGAACTCCG GCCATCTGTC ACAGTACGTA TATGACCAGA TCGGCACCATTTGTCTCGGC CTGACAATCT CGCGCGCCAT TGGCCATGCA AAGCTGTCCT GCCGTTCGGAGAGACTAGAG AGCCAGTTGG CAAATTGACA TTTGCGATAG GTGGGGCGGC TTTGACTATGACATGATGAC AGATCCAGAT GGTCCTCCGC TAGTCCCCCC GAGCCCGAGG ACAGCACACTAGCTCACACG AACTGACAGC GCGGAGGAGG ACACGTACCG GGATGACACC GCCACCCATTTGCTGGCAAG CCGGGGTGCG CCGGCGGTTC AGGTTGAATC CTTCCTAATG GTCGTGCTAGCAAACCCCGC AAGCTCAGTG CGGGTCCAAA ACCCATTAAT TATCCCACAA AGCCGCCGTTAGACGTAGAA TCGACGCCGC GCGCCACGGC CGGCGGCGGC TACCTGGCTC TTACCACCATCATTCGCTTG TCCGTTCCGT CGCCCCCGCC ACCCTCTCAG AGATGGAGGC GGTTAAGTGCCTGTCGACTA TTGCAGAACG TCGTCAGGCT CGCTAGTTCG ACCGAGCATC CTAGATACATAATCCAAATT CCGCTCGGCG ATTATAGGAG GGTGATAGTA CTGAGTACAG GGCGAAAAACGTTGAAAAGG TCAGCGAGGC CCCCACATGT CTCCCCCGGT CGCGTTCGCA TTCAACACCCTCTGCGCTGC GTTTCATGGA AGTTTCCAGC AGCCACGCCC ACGCGCATGG ACGCGGCTGATCTTATAAAG GTGGCGCGCG TCCCAACCTC GGGAGCCATC ATTTCACCAG AAGCTGCAAATTGCAAGCTC TCCTCCCTAG CTAGCCTCTC CAGCAGCCCA ACCACAGCCT GCAGCTGCAGCTCGCGTTGG CACAGCGCCG CCTGAACGCG TGCTAATTTA AGCTCTGTCG TAGCTCAACGCGGCCGCCGG GCTTTCGCCG ACGACGTCAA AATGGCCGGT GGTAGCAGTG CCGAGCAGCTCCTATCCAGG ATCGCCTCCG GCGATGGCCA CGGCGAGAAC TCGTCCTACT TCGACGGGTGGAAGGCCTAC GACATGGACC CTTTCGACCT GCGCCACAAC CGCGACGGCG TCATCCAGATGGGCCTCGCC GAGAACCAAG TACGTGACGT AGCCCTGCCG CATGCAGCTA CAGCTACACCCTTTCGACCT GCGCAACAAC CGCGACGGCG TCATCCAGAT GGGCCTGCTG TCGATGGAATGCTCATGTAA TTAAACCACC GGCCGGGGCG TGTTTTGCAG CTGTCCCTGG ACCTGATCGAGCAATGGAGC ATGGAGCACC CGGAGGCGTC CATCTGCACG GCGCAGGGAG CGTCGCAGTTCAGGAGGATA GCCAACTTCC AGGACTACCA CGGCCTGCCG GAGTTCAGAG AGGTATTAATTAAGTTAACT AACAGCTCGG CTAAGGAAAC GCCAGAATCA TTGATTAGGT TTGCTGCTCTCTAATGGCGA CTGCGAAAAC GACGGAGCAG CTACCGGCCA GCCGGCCGGC GGTTAGCTAGCACTAGCAGC CGCCTTCCTG ACAGATCATC CATGACGTTT TGATTGTTGC AGGCGATGGCCAAGTTCATG GGCCAGGTCA GGGCCGGGAA GGTGACGTTC GACCCCGACC GCGTCGTCATGTGCGGAGGC GCCACCGGCG CGCAGGACAC TCTCGCCTTC TGCCTCGCTG ACCCGGGCGACGCCTACCTC GTGCCGACGC CATACTACCC AGCGTATGTC TCGACCAACG TCATCCTTGTACTTGTACCA AAATTAGTCA CCCGTTGACA CCAAAGTTGG TAAGAGGGTA AGAGCAGGGAAAGGCAGAGC TAAGGCCCTG TTTGGTTTGA GGTGACTAAA GTTTAGTGAC TAATATTTAGTCACTTTTAG TCTCTAAAGA AGTAAACATG GTGACTAAAG TGAAGTGACT AAATTTTAGTTCTTTAGTCA CTAAGAGGCT GACTAAAAGG GACTAAAGTA GTATTTTTAC CTTATTTGTCCTCTCCACTT TCTTCTTATA GCAAACATCT ATTAATTAAT AGGGATAAAA TAATCATTATTCACAGCAAT TAATGCCCTT TAGTCCGGTT TAGTCACTGG AACCAAACGG GATACTTTAGCGACTAAACT TTAGTCACTA AAATTTAGTC TAGTGACTAA GGGAACCAAA CAGGACCTAATTCGAGTGTG ATGTCAACAA GACAACAAAT AATAGCCAAT TGTAGCCCCT CGCCATCTTTCCTTGTTTGG GTAACGTTTC AAAATTTAGG GGGTGTTTGG TTTCTAGCGA CTAATGTTTAGTCCCTTCAT TTTATTCCAT TTTAGTATAT AAATTGTCAA ATATAAAAAC CAAAATAGAGTTTTAGTTTC TATATTTGAC AATTTTACAA CTAAAATGAA ATAAAATGTA GGGACTAAAGTATAAACTAA ACACCCCCTT ACCTCGATCA CGAACCTCTA AAAGTAAGTA GCACCCTCCTCCCCCACAGT CAAATCAACA TAATACAGTA CAATAGACCT TGTTAGTCGC ATGGATGATTGTCGTCAAGT GGGCAACGCA ATCTAGTCAC GTAAGGAAAA CCATGCACGT TGTTCATACACGGTCTGTTT CCATGCGACT TTAATTTCCA CGCACGTTTG CATCGTTGAC CAACCAACTGAACGTGCCTG TAGGTCCCGC ACAGCAACGT AAGCATATGC ATGCACGTAC GACGTACGGCACGGGAAAAA AATTCTGCAC ACCGTATTTT ACAGCTCTTC ATATCCACCA CATGTAGCGGCCCCACAAAA AACAGATTAA AATTTGCAAC TTAATCCTTA AGTAATTTGT TTTTCTTCTATTTATATAGA TTATCAGTTG ATGGATGTGT GAAGTTGTAA AAGAGATTAT TTGTATCCAGGATTAAAATA ATTTTCCGTA CGGCACGCCT GCAGTACTCA TTCTCGCCAG CCCTGAGCCCCTGATATATG ACACGCTTTT CATTGTTCAC ACAGTTTCGA CCGTGACTGT TGCTGGAGGTCAGGCGTGAA GCTGCTGCCC ATCGAATGCC ACAGCTCAAA CAACTTCACC CTCACACGGGAGGCGCTCGT GTCGGCCTAC GACGGCGCGC GGAGGCAGGG CGTCCGCGTC AAGGGCGTCCTCATCACCAA CCCCTCCAAC CCGCTGGGCA CCACCATGGA CCGCGCCACG CTGGCGATGCTCGCCAGGTT CGCCACGGAG CACCGTGTCC ACCTCATCTG CGACGAGATC TACGCGGGCTCCGTCTTCGC CAAGCCGGAC TTCGTGAGCA TCGCCGAGGT CATCGAGCGC GACGTCCCGGGCTGCAACAG GGACCTCATC CACATCGCGT ACAGCCTCTC CAAGGACTTC GGCCTCCCGGGCTTCCGCGT CGGCATCGTC TACTCGTACA ACGACGACGT CGTGGCCTGC GCGCGCAAGATGTCCAGCTT CGGCCTCGTC TCCTCGCAGA CGCAGCACTT CCTGCCCAAG ATGCTGTCGGACGCGGAGTT CATGGCCCGC TTCCTCGCGG AGAGCGCGCG GCGGCTGGCG GCGCGCCACGACCGCTTCGT CGCGGGACTC CGCGAGGTCG GCATCGCGTG CCTGCCCGGC AACGCGGGGCTCTTCTCGTG GATGGACCTG CGGGGCATGC TCCGGGACAA GACGCACGAC GCGGAGCTGGAGCTGTGGCG GGTCATCCTA CACAAGGTGA AGCTCAACGT GTCGCCCGGC ACGTCGTTCCACTGCAACGA GCCCGGCTGG TTCCGCGTCT GCCACGCTAA CATGGACGAC GAGACCATGGAGGTCGCGCT CGACAGGATC CGCCGCTTCG TGCGCCAGCA CCAGCACAAG GCCAAGGCCGAGCGCTGGGC GGCCACGCGG CCCATGCGCC TCAGCTTGCC GCGCCGGGGA GGCGCCACCGCTTCGCACCT CCCCATCTCC AGCCCCATGG CGTTGCTGTC GCCGCAGTCC CCGATGGTTCACGCCAGCTA GTCACCGAGC ATCCGGCAAG ACTGGCTGTA GGGTGTGCCC GTACATCCGTACGTACACCT TTTTTTCCCA TTCACGTGAC TGCAATCAAG TCTATGGGAT GGTTGACAAAAGACTATCTA GACAAGAGTG GGCGTAGTAC GTAACTAGTT TGACGTTGTA CAGGCGTCAGCAGGTATCGG TAAGCAGCTA GTCAAAAGCA CGCAAGCAGG ACGCATTTGT CCTCGATACTTTCGTGTAAA TCTCTCTCTA TTTTTTTTTG CGAAATTCGC GTGTATGGTT TGTTTTGACGTTGGTATAAA GTATGGTAGA ATAACGATGG GAAATGGCAA TTTAGTCCTC CCGATCAATTGTTATTGTAA ACCACTGACG AAAGTTAAGA ACAGAAGCTG TACCAGAAGG GTGAATAAAAATACCACATA GGTATTGAAT TAATAATCTA TGTATTTCGA GTTACTCCTG CAAGATATCTATTTTTTCAT GCTGTGCTGG CCACATTTGC CTCTTCTTCA AACTAGTTTC TCGCA SEQ ID: 2Gene ACS6 (ACC6) CGGCTAGTTT TGATAGTTAG ACGATGTTCT GACAGCGCAC CAGACAGTAAACC Synthase gene CCAGTGACAG TCCGGTGCCT GGCTAAATAT CGAGCCAGCG AACAGCCCGCsequence (A65) TCTCGGGTTT CTACGGGGGC AGAGGGTTGC TCTCGGGGCA TTCTTGTGCTfrom Zea mays (Zm) CACTGTCAGG GGGAGCACCA GACAGTCCGG TGCACAGCGAACAGTCTCAT inbred ‘B73’ (= GCCCCTAGGT CAGCAAGTCA AAGTTCTCTT CCTTAGATTTTTCTAAACCG ACC6 pcr fragment TTTTCGTTTT AACTTGTGAG TGAGTTATCG AGTGACACCTAGCACTAGTT originally GTGAGTATGA ACACCAACAC TATATTACAT TTCTCTTGGTCAAACTACTC isolated from ATCCACAACC ACTCTTTATA GTACGGCTAA AATAAAAATAGAAGTCCTAA inbred ‘Oh43’) CTTTATACCA AGTGTCAACA ACTCCTTCGG ACACTTAGAATATAAAGTCC TTCATCTTTT GTTTCGCCTT TTTCCGCCCT CGCTTCAAGT TCTCATCCGAGGGATTGTTT TATCGTTGTA GTGCAACTTC ATGCAATGTG ACCTAACTTG CCATTTGCTCTTCAAAACAC ACGTTAGTCA TATAATATTA CGTTGTCATT AATCTCTATC GATATTTTTCACCCATTACG TTGTCACTAG ATGCTTTCAC CCATTTCGAT TTCAGACGAT GTCTTCGGACGTTGCGGGCC ATGTGTCCAA ACGTGGTTAA GTGTGGTCGG GAAATACCCG ATCGAGGTTGAGTTCGGCCT TCGCTCCGAC ACCCAGCCGT GTCATTACTG TCATATATAT TGTAGCAATGTCAAAAAAAA TCAAAACATT GAGTATGACG TATAGGGCAC ATATGTCATT AAACTTATTCAGTGTAATGA TATATTATCA TCACGGGACT TTTTTTTAAT GTATGTATTA GATTACCTCTGCCATGCACT ATACAAACAG CTACGCCGCA GTCGCAAGCA AACAGGCTCT AAAAGGCTTCAGTCGGAGAA GGATATGAGA GCGGTGAGTA CCAAACGGGT ATCTTCCCCT TCCAAATGATATAAGCCTAC TTGTTTGACC CCAGCCCGCA GGCAGTCATC TGCTATAATA GGCTAATACAACTTGTGTAC TCTAGTCTGC TCTCGCCGCG TTGTCCGCAT GCTGAACCCG CGATGTTAACACCTCCCTGA ACGAGTCCTC TGTTCCTCAA CTGAAATTCA GCAATAAAAG GAAAAATCCGCGGTCCCTGT CCCTGTCCAG CACCGCACTC TCGCACTTGT GCTGCAGGCT TCTGAGCTCGGCACCTGCTG CTAGCTGCTG CTATATATAG ACGCGTTTTG GGGTCACCAA AACCACCAGCTGATCAACAG CTAGCTTCAT TCCTCTGCCT CTCTCTCCCT CCTTCGCCAA CTGGCCATCTCTGTTGTCTC TCGCTAGCTA GCTCGCTCGC TCGCTCGCCA GTCACCACAC ACACACACACACACTGTGTG TCTGTGCCTG ACGCCGCCCC CCAGTTTCAA ACGAACGACC CAGCCAGAAACGCGCGCGCG CCAAAGCTAC GTGAGTGACG TGGCAGCATG GTGAGCATGA TCGCCGACGAGAAGCCGCAG CCGCAGCTGC TGTCCAAGAA GGCCGCCTGC AACAGCCACG GCCAGGACTCGTCCTACTTC CTGGGGTGGG AGGAGTATGA GAAAAACCCA TACGACCCCG TCGCCAACCCCGGCGGCATC ATCCAGATGG GCCTCGCCGA GAACCAGCTG TCCTTCGACC TGCTGGAGGCGTGGCTGGAG GCCAACCCGG ACGCGCTCGG CCTCCGCCGG GGAGGCGCCT CTGTATTCCGCGAGCTCGCG CTCTTCCAGG ACTACCACGG CATGCCGGCC TTCAAGAATG TGAGTGCCTGCTAGCTTACT CATTCCCAGG CAGGCAGGCA GCCAGCCACG GCATGCCGAA CCAGTCTCACCTCTCTCGCG CACATGAATG CGTGATTCCC GCAGGCATTG GCGAGGTTCA TGTCGGAGCAACGTGGGTAC CGGGTGACCT TCGACCCCAG CAACATCGTG CTCACCGCCG GAGCCACCTCGGCCAACGAG GCCCTCATGT TCTGCCTCGC CGACCACGGA GACGCCTTTC TCATCCCCACGCCATACTAC CCAGGGTATG TGTGTGTGTT GCCTTGTACT TACTCGTCGC CGCAAGTACTTGCAGTAGGG AACGTGCAAG TGGCGGCGGG GCGGCGTCTG GGTGTCGCCG CGATGCACGTTACTGCTATT AAAGTAGTAG TAGTACACTA ATAGCTAGGC CCACCACAGC ACACGATGACATGACGAACG ATGGATGGGA ACGGCTGCTG ACTGGGCCTG CTTGCTCTTG TCTGCAGGTTCGACCGTGAC CTCAAGTCGC GCACCGGCGC GGAGATCGTC CCCGTCCACT GCACGAGCGGCAACGGCTTC CGGCTGACGC CCGCCGCGCT GGACGACGCC TACCGGCGCG CGCAGAAGCTGCGGCTGCGC GTCAAGGGCG TGCTCATCAC CAACCCTTCC AACCCGCTGG GCACCACGTCGCCGCGCGCC GACCTGGAGA TGCTGGTGGA CTTCGTGGCC GCCAAGGGCA TCCACCTGGTGAGCGACGAG ATATACTCGG GCACGGTCTT CGCGGACCCG GGCTTCGTGA GCGTCCTCGAGGTGGTGGCC GCGCGCGCCG CCACGGACGA CGGCGTCGTC GGCGTTGGGC CGCTGTCGGACCGCGTGCAC GTGGTGTACA GCCTGTCCAA GGACCTGGGC CTCCCGGGGT TCCGCGTGGGCGCCATCTAC TCGTCCAACG CCGGCGTGGT CTCCGCGGCC ACCAAGATGT CGAGCTTCGGCCTGGTGTCG TCCCAGACGC AGCACCTCCT GGCGTCGCTC CTGGGCGACA GGGACTTCACGCGGAGGTAC ATCGCGGAGA ACACGCGGCG GATCAGGGAG CGGCGCGAGC AGCTGGCGGAGGGCCTGGCG GCCGTGGGCA TCGAGTGCCT GGAGAGCAAC GCGGGGCTCT TCTGCTGGGTCAACATGCGG CGCCTCATGC GGAGCCGGTC GTTCGAGGGC GAGATGGAGC TGTGGAAGAAGGTGGTCTTC GAGGTGGGGC TCAACATCTC CCCGGGCTCC TCCTGCCACT GCCGGGAGCCCGGCTGGTTC CGCGTCTGCT TCGCCAACAT GTCCGCCAAG ACGCTCGACG TCGCGCTCCAGCGCCTGGGC GCCTTCGCGG AGGCCGCCAC CGCGGGGCGC CGCGTGCTTG CCCCCGCCAGGAGCATCAGC CTCCCGGTCC GCTTCAGCTG GGCTAACCGC CTCACCCCGG GCTCCGCCGCCGACCGGAAG GCCGAGCGGT AGCCGGTCCC CGTCCGCGCC GACCGCACGT GCTCAGCTCAGCAGCTTCAC AGCTCACCAC CAGTCACCAC CACCACCACC ACCACCACCT GGGGTGGAGGCGTCGAGCAA GCAATGTTCA TAGAAACCAC GGTCACGTAC TATACAATAC TACTACCGTACCACACCACA CGGCAGCATC ATTAGCAGTA GGAGATTAGT AGTAATCATT AATTCCTTATTGGGTTCTTG TAATTTCGTA TATACCACGC CGCCATTTTT CCTTGGGGCC AGGCCAGCCGATAGGTGCCC GAGGGCCACT GCACTGCACT GCTGTATTAG GTAGGACCAG GAGTCGTGGGTAGCGAATCC ACCTTCCAGC AGCAGGCATC ACATTTGTGT ATTTTTCGAC TGGGTCTCCCGGTTGTTTT SEQ ID: 3 Gene ACS7 (ACC7) GCTGGTAGCT TCTTTAACTG ATCTCAATGGGGCATTTCGG TGGCTAGCAA ACC Synthase gene TTCACATTAA TAATTTAAAA GTGAATTTCAGGTGTACATT TGATGGCCTC sequence (A50) CGATATGGTG CAGCCTTCAA TCCTCTACAATGTGCGAGAA TGTTGCTCCG from Zea mays (Zm) GAGGGTAGAG GCGATTAACGGCTGAACACA GATGACCTCC TCGGAGTCAT inbred ‘B73’ (= GTTTCTAATT ATCTACACTACGATTCTCTT TCCGTTGATA AAATATTTGT ACC7 pcr fragment TTTATTGTCC TGTGAGCTAATGATAACATT GATGGTAAGT AAATATAGTC originally CATGCATATT CTCATCACAGATGGCTGAAA AACTCCCCGT GCTGCTACAC isolated from TACTAGAGTC TTCATGTGCATACTTACTTC AAGAACTCAA GGTACACAAA inbred ‘Oh43’) GTTTTCTCAA CAGAAGAATGTGTATCTGTT TGATTCCAGC TGAAATGCTT ACTAAACTCA GTGTGTCGCT TTAGATGATATGAGATGAAG TTGGGCAAGA CCAAAGTGAA AGGGAGAGAA TAACGGAAGA ACTTGTTCGCCAACTTGGAG AAACCAATAC TAAAACTCAG TGAATATATG TGTGGATTTG GAAGCAAGTGAATTTTACAG AAAAGTTTTT TGAGAGTGTT TATATGAATC GTACTCATCT GTTTATTTTGATGACTGCAA TATAACTACT TGTATTTATA GTTTGAGATC AAGAAAATAA GTTATTATTTAGAAATAATA AAAAATTATA GTGATGTTTG TTGTTCCGTA TCAATGTTTC ATACAAATGTTTTACTTCCG TCGCAACACA CGGGAATATA CCTATAATAT ATATTGTTAT CATGTTATTATACGGTTCCG TTGCAACGCA CGGGCACATA CCTAGTACAA AAATAATTAC GCATCCCGCAGTTGACATCT GGGAGCGCTA CAAATAATGA AGGCAGCTGG TCCACCACAC GAACTGACAGCGCGCAGAAG GGAGTGCACC GGCCCACCGG GATGGCACCG CGAATCAGCC TCGGCAGCGCCATACTGCCC ACCCATTTTT TCTGGCGAAT CCGGGTGCGG CGGGCGGTTG AGGATGAATTGAATAATACT CTACTTCCTA ATGGTCGTGC TAGCAGACCC TGGAAGCTCA GTGTGGCTCCAAAACCCATT AATTAATTAA ACCACAAAGC CGCCGCCGTT AGACCTAGAA CCACCGCTGCGCTCGCCGGG CGCCGGCTAC CCGGCGTAAC TGCCGTCACC ATCCACCACC TGGCCGCTCCGTTCTTTCCT CCACCCCAAG ATGGACCCGG TTAACCTGTC CAATCTTACC TCATATGCGTAATCAACTAT TTTAACTTTC ACTATATATA TATGTTAATA TTTATAATAT ATAATTTGTAGTATAAGATA AATATTTGAA TTTGTTTTTA TAATAAACGT ATTTTGACAT ATAAATATTCGTAATATTTT TTTTTTACAA ATCTGACTAG ATTTTAAATC TGTAACGAGG AGTACATAGTACGAAATGTT GAAAAGTCAG CGTGTCTTTG GTCGCGTTCG CATTCATTCT TTCTTTACCTCAGCCACCCA CCTGCCACAC CCTGTGGGCC GTGGCGCCTT CACGGAAGGT TCGCCGGCCACGCATGGAGG CGGCTCTTTA TAAAGCTGGT GCGCGGGCGG GAGGGGAGAG GGCACCAGAAGCAGCCAGCA AGCTCATGCC CTTCAAAAGC CTCCGGCAGC CCAGCGCCCC AGCCAGCTAGTGGTGATCTC TCATCTCAGC AGCGCGCCTG AACGTGTGCT CCCTGCTAAC CTCTGCGCCTCGATAGGCAA AGGAAAATCA AACCGATCGT CGTCAGATTA AATGGCCGGT AGCAGCGCGGAGCAGCTCCT CTCCAGGATC GCCGCCGGCG ACGGCCACGG CGAGAACTCG TCCTACTTCGACGGGTGGAA GGCCTACGAC ATGAACCCTT TCGACCTGCG CCACAACCGC GACGGCGTCATCCAGATGGG CCTCGCCGAG AACCAAGTAC GTACCTATAG CGTGTACCTA CCCTTCCGATCTGTAGTACT GCCCACACTT GCTGCATGCT GCTGCCGATC CAAGTCCAAT GCTCATGTAAACTGGCGTGC TGCAGCTGTC GTTGGACCTG ATCGAGCAAT GGAGCGTGGA CCACCCGGAGGCGTCCATCT GCACGGCGCA GGGCGCGCCG CAGTTCCGGA GGATAGCCAA CTTCCAGGACTACCACGGCC TGCCGGAGTT CAGAGAGGTA ACTAACTAGT AGTGATTAAC AAGCAAATAAACGCCAGGAT CACTGCATCG ATTAGCTAGG TTTGCTGCTG CTGCTGCTGC TGTCTAATATAATGGCGACT GCACGCGAAA AGCGACGGAG CAGCTACCGG CCGGCGGCTA GCTAGCTAGCTGGCACTGGC AGCGCAGTCG CCTTCATGAG TCCACGCACG CGCGGCTACG TCTTAATGATCGATCGGCTC GTCGTTTGTT GCAGGCGATG GCCAAGTTCA TGGGGCAGGT GAGGGGCGGCAAGGTCACGT TCGACCCCGA CCGCGTCGTC ATGTGCGGAG GAGCCACCGG CGCGCAGGACACTCTCGCCT TCTGCCTCGC TGACCCGGGC GACGCCTACC TCGTGCCGAC GCCTTATTACCCAGCGTATG TTCTGACGTC ACCCTTGTAC TGCCAAACTA CTACTCAGGT CCTAGTCATATCCGTAGACA CGAAAGGGTC GGTGGGTCTG GGTTGTTGGT TGGTCAAGAG CACGCAAAATTGAGCTAATT CGACTACGTA CGTGTCAATG TCAACTAGCC ACTTATCTTT CCTTGTTTGGGTAAAGTTTC AAAACTTATT AACTCGATCA GGAACCTCTC TAAAAAGCAT TCACCTATTTTTCCCCCGTA AGGCGGTAAC CAAATCTAAA CGATATACCC TTGTTAGTCG CACTGATGACTGCATTGTCG TCAAGTGGAC AACGCAATCT AGTCACGCGA CCTCTAAGGA AAACCACGCACGTATACGCA CTTCGTGCAC GGTCTGTTCC ACGCGACTTT AGTTTCCATG CACGTTTACATCGTTGACCA TCCGCAGTCC GCACAGCAAC GTAAGCATAA ACATGCACGC ACGACGTACGGCACACCGTA CCTGTTCCTC TCGAGGGCTG AGACCCTGAC ACGTTTTTTT CGTTGTGTGGTGATCACAGT TTCGACCGCG ACTGTTGCTG GAGGTCAGGA GTGAAGCTGC TGCCCATCGAATGCCACAGC TCGAACAACT TCACCCTCAC CAGGGAGGCG CTCGTGTCGG CCTACGACGGCGCGCGGAGG CAGGGCGTCC GCGTCAGGGG CATCCTCATC ACCAACCCCT CCAACCCGCTGGGCACCACC ATGGACCGCG GCACGCTGGC GATGCTCGCC GCGTTCGCCA CAGAGCGCCGCGTCCACCTC ATCTGCGACG AGATCTACGC GGGCTCCGTC TTCGCCAAGC CGGGCTTCGTGAGCATCGCC GAGGTCATCG AGCGCGGCGA CGCCCCGGGC TGCAACAGGG ACCTCGTCCACATCGCGTAC AGCCTCTCCA AGGACTTCGG CCTCCCGGGC TTCCGCGTCG GCATCGTCTACTCCTACAAC GACGACGTGG TGGCCTGCGC GCGCAAGATG TCCAGCTTCG GCCTCGTCTCGTCGCAGACG CAGCACTTCC TGGCGATGAT GCTCGCCGAC GCGGAGTTCA TGCCACGCTTCCTCGCGGAG AGCGCGCGGC GGCTGGCGGC GCGCCACGAC CGCTTCGTCG CGGGCCTCCGCGAGGTCGGC ATCGCGTGCC TGCCGGGCAA CGCGGGCCTC TTCTCGTGGA TGGACCTGCGGGGCATGCTC CGGGAGAAGA CGCACGACGC GGAGCTCGAG CTGTGGCGGG TCATCGTACACAGGGTGAAG CTCAACGTGT CGCCCGGCAC GTCGTTCCAC TGCAACGAGC CCGGCTGGTTCCGCGTCTGC TACGCCAACA TGGACGACGA CACCATGGAG GTCGCGCTCG ACCGGATCCGCCGCTTCGTG CGCCAGCACC AGCACAGCAA GGCCAAGGCC GAGCGCTGGG CGGCCACGCGGCCCCTTCGC CTCAGCTTGC CGCGCCGGGG AGCAACCACC GCTTCGCATC TCGCCATCTCCAGCCCCTTG GCGTTGCTGT CGCCGCAGTC CCCGATGGTC CACGCCAGCT AGGTAGTCACCGAGCGTTCG GTAAGACTGG CTGTAGGTTG TGCCCTCACA TGACTGCAAA CAAGTGGACAAAAAAAAAGA CAAGACTAAT AAAGGGCGTA CGTAGCTAGC TTGACATTAC ACAGAGTGACAGAGACGTTG CACAGGCGTC AGCAGGCGTC GGCGGTAAGC AGCTAGTCAA GTAGGACGCATTTGTCCTCG ATTTTTTCGT GTTTTTTTTT TGACGAAGGG GCGAAGCCCC CTATTTCATTAAGAAATAGG AAAGTATGAA ACAACCGCAC CCACGCGGTA GGACCTCCAA AAAGAACAGCCACGGCCAGA AAGTAATCTA GACTCTAAAC ACTATCGCTA GATCAGTGAA GAGACTATGATAACAGGGAA AGTTTTGGCC TACGAAGAGC TACATAAGAC TTTCTTATAT ACAACCAACCAAGACAGGCA GAAGCCACAA AAGACCTGAA CAGAATGGCC AACAAAAGAC AGACAACTATCCCAACAAGG TTTCACAGCT TCAGCATCTT TGTCATCCAG AAATCCGCCT GTCAAGAGGACACCACCCCA AGGCCCTCCC GAAAGCTTCA CTTGCCGTCT TTCGGATTAA CCTGCTTCCTAGCACCACCA TTCTTTGCTC CTTCTTTTTC TGACGAATCG CCCAAGAATC CAACCAGAAGCAGCAAAGAA AAATGATGTT AGATGGGTCA AGTAAATGAC TATTCCCAAA ACACCAATCATTCCTAGTGC GCCAAATAGC CCAGAATAAA GCACCACAAC CAAATAACAC CAACTGAGCCATCGTGTCTT TTGGTTTACA AAACCAATTG TCATACAAAT CTTTGATATT TTTTGGAATAGATCTCAAAT TCAGGGCCAC TTGAATAACT CTCCACATGT ATTGAGCAAT GGGGCAATAG AAAAASEQ ID: 4 cDNA ACS2 (ACC2)ATGGCCGGTGGTAGCAGTGCCGAGCAGCTCCTATCCAGGATCGCCTCCGGCGAT ACC Synthase cDNAGGCCACGGCGAGAACTCGTCCTACTTCGACGGGTGGAAGGCCTACGACATGGAC sequence (A47)CCTTTCGACCTGCGCCACAACCGCGACGGCGTCATCCAGATGGGCCTCGCCGAG from Zea mays(Zm) AACCAACTGTCCCTGGACCTGATCGAGCAATGGAGCATGGAGCACCCGGAGGCG inbred‘B73’ (= TCCATCTGCACGGCGCAGGGAGCGTCGCAGTTCAGGAGGATAGCCAACTTCCAG ACC 2pcr fragment GACTACCACGGCCTGCCGGAGTTCAGAGAGGCGATGGCCAAGTTCATGGGCCAGoriginally GTGAGGGCCGGGAAGGTGACGTTCGACCCCGACCGCGTCGTCATGTGCGGAGGCisolated from GCCACCGGCGCGCAGGACACTCTCGCCTTCTGCCTCGCTGACCCGGGCGACGCCinbred ‘Oh43’) TACCTCGTGCCGACGCCATACTACCCAGCGTTCGACCGTGACTGTTGCTGGAGGTCAGGCGTGAAGCTGCTGCCCATCGAATGCCACAGCTCAAACAACTTCACCCTCACACGGGAGGCGCTCGTGTCGGCCTACGACGGCGCGCGGAGGCAGGGCGTCCGCGTCAAGGGCGTCCTCATCACCAACCCCTCCAACCCGCTGGGCACCACCATGGACCGCGCCACGCTGGCGATGCTCGCCAGGTTCGCCACGGAGCACCGTGTCCACCTCATCTGCGACGAGATCTACGCGGGCTCCGTCTTCGCCAAGCCGGACTTCGTGAGCATCGCCGAGGTCATCGAGCGCGACGTCCCGGGCTGCAACAGGGACCTCATCCACATCGCGTACAGCCTCTCCAAGGACTTCGGCCTCCCGGGCTTCCGCGTCGGCATCGTCTACTCGTACAACGACGACGTCGTGGCCTGCGCGCGCAAGATGTCCAGCTTCGGCCTCGTCTCCTCGCAGACGCAGCACTTCCTGGCGAAGATGCTGTCGGACGCGGAGTTCATGGCCCGCTTCCTCGCGGAGAGCGCGCGGCGGCTGGCGGCGCGCCACGACCGCTTCGTCGCGGGACTCCGCGAGGTCGGCATCGCGTGCCTGCCCGGCAACGCGGGGCTCTTCTCGTGGATGGACCTGCGGGGCATGCTCCGGGACAAGACGCACGACGCGGAGCTGGAGCTGTGGCGGGTCATCGTACACAAGGTGAAGCTCAACGTGTCGCCCGGCACGTCGTTCCACTGCAACGAGCCCGGCTGGTTCCGCGTCTGCCACGCTAACATGGACGACGAGACCATGGAGGTCGCGCTCGACAGGATCCGCCGCTTCGTGCGCCAGCACCAGCACAAGGCCAAGGCCGAGCGCTGGGCGGCCACGCGGCCCATGCGCCTCAGCTTGCCGCGCCGGGGAGGCGCCACCGCTTCGCACCTCCCCATCTCCAGCCCCATGGCGTTGCTGTCGCCGCAGTCCCCGATGGTTCACGCCAGC SEQ ID: 5 cDNA ACS6(ACC6) ATGATCGCCGACGAGAAGCCGCAGCCGCAGCTGCTCTCCAAGAAGGCCGCCTGC ACCSynthase cDNA AACAGCCACGGCCAGGACTCGTCCTACTTCCTGGGGTGGGAGGAGTATGAGAAAsequence (A65) AACCCATACGACCCCGTCGCCAACCCCGGCGGCATCATCCAGATGGGCCTCGCCfrom Zea mays (Zm)GAGAACCAGCTGTCCTTCGACCTGCTGGAGGCGTGGCTGGAGGCCAACCCGGAC inbred ‘B73’ (=GCGCTCGGCCTCCGCCGGGGAGGCGCCTCTGTATTCCGCGAGCTCGCGCTCTTC ACC6 per fragmentCAGGACTACCACGGCATGCCGGCCTTCAAGAATGCATTGGCGAGGTTCATGTCG originallyGAGCAACGTGGGTACCGGGTGACCTTCGACCCCAGCAACATCGTGCTCACCGCC isolated fromGGAGCCACCTCGGCCAACGAGGCCCTCATGTTCTGCCTCGCCGACCACGGAGAC inbred ‘Oh43’)GCCTTTCTCATCCCCACGCCATACTACCCAGGGTTCGACCGTGACCTCAAGTGGCGCACCGGCGCGGAGATCGTCCCCGTGCACTGCACGAGCGGCAACGGCTTCCGGCTGACGCGCGCCGCGCTGGACGACGCGTACCGGCGCGCGCAGAAGCTGCGGCTGCGCGTCAAGGGCGTGCTCATCACCAACCCTTCCAACCCGCTGGGCACCACGTCGCCGCGCGCCGACCTGGAGATGCTGGTGGACTTCGTGGCCGCCAAGGGCATCCACCTGGTGAGCGACGAGATATACTCGGGCACGGTCTTCGCGGACCCGGGCTTCGTGAGCGTCCTCGAGGTGGTGGCCGCGCGCGCCGCCACGGACGACGGCGTCGTCGGCGTTGGGCCGCTGTCGGACCGCGTGCACGTGGTGTACAGCCTGTCCAAGGACCTGGGCCTCCCGGGGTTCCGCGTGGGCGCCATCTACTCGTCCAACGCCGGCGTGGTCTCCGCGGCCACCAAGATGTCGAGCTTCGGCCTGGTGTCGTCCCAGACGCAGCACCTCCTGGCGTCGCTCCTGGGCGACAGGGACTTCACGCGGAGGTACATCGCGGAGAACACGCGGCGGATCAGGGAGCGGCGCGAGCAGCTGGCGGAGGGCCTGGCGGCCGTGGGCATCGAGTGCCTGGAGAGCAACGCGGGGCTCTTCTGCTGGGTCAACATGCGGCGCCTGATGCGGAGCCGGTCGTTCGAGGGCGAGATGGAGCTGTGGAAGAAGGTGGTCTTCGAGGTGGGGCTCAACATCTCCCCGGGCTCCTCCTGCCACTGCCGGGAGCCCGGCTGGTTCCGCGTCTGCTTCGCCAACATGTCCGCCAAGACGCTCGACGTCGCGCTCCAGCGCCTGGGCGCCTTCGCGGAGGCCGCCACCGCGGGGCGCCGCGTGCTTGCCCCCGCCAGGAGCATCAGCCTCCCGGTCCGCTTCAGCTGGGCTAACCGCCTCACCCCGGGCTCCGCCGCCGACCGGAAGGCCGAGCGG SEQ ID: 6 cDNA ACS7 (ACC7)ATGGCCGGTAGCAGCGCGGAGCAGCTCCTCTCCAGGATCGCCGCCGGCGACGGC ACC Synthase cDNACACGGCGAGAACTCGTCCTACTTCGACGGGTGGAAGGCCTACGACATGAACCCT sequence (A50)TTCGACCTGCGCCACAACCGCGACGGCGTCATCCAGATGGGCCTCGCCGAGAAC from Zea mays(Zm) CAACTGTCGTTGGACCTGATCGAGCAATGGAGCGTGGACCACCCGGAGGCGTCC inbred‘B73’ (= ATCTGCACGGCGCAGGGCGCGCCGCAGTTCCGGAGGATAGCCAACTTCCAGGAC ACC7 perfragment TACCACGGCCTGCCGGAGTTCAGAGAGGCGATGGCCAAGTTCATGGGGCAGGTGoriginally AGGGGCGGCAAGGTGACGTTCGACCCCGACCGCGTCGTCATGTGCGGAGGAGCCisolated from ACCGGCGCGCAGGACACTCTCGCCTTCTGCCTCGCTGACCCGGGCGACGCCTACinbred ‘Oh43’) CTCGTGCCGACGCCTTATTACCCAGCGTTCGACCGCGACTGTTGCTGGAGGTCAGGAGTGAAGCTGCTGCCCATCGAATGCCACAGCTCGAACAACTTCACCCTCACCAGGGAGGCGCTCGTGTCGGCCTACGACGGCGCGCGGAGGCAGGGCGTCCGCGTCAGGGGCATCCTCATCACCAACCCCTCCAACCCGCTGGGCACCACCATGGACCGCGGCACGCTGGCGATGCTCGCCGCGTTCGCCACAGAGCGCCGCGTCCACCTCATCTGCGACGAGATCTACGCGGGCTCCGTCTTCGCCAAGCCGGGCTTCGTGAGCATCGCCGAGGTCATCGAGCGCGGCGACGCCCCGGGCTGCAACAGGGACCTCGTCCACATCGCGTACAGCCTCTCCAAGGACTTCGGCCTCCCGGGCTTCCGCGTCGGCATCGTCTACTCCTACAACGACGACGTGGTGGCCTGCGCGCGCAAGATGTCCAGCTTCGGCCTCGTCTCGTCGCAGACGCAGCACTTCCTGGCGATGATGCTCGCCGACGCGGAGTTCATGGCACGCTTCCTCGCGGAGAGCGCGCGGCGGCTGGCGGCGCGCCACGACCGCTTCGTCGCGGGCCTCCGCGAGGTCGGCATCGCGTGCCTGCCGGGCAACGCGCGCCTCTTCTCGTGGATGGACCTGCGGGGCATGCTCCGGGAGAAGACGCACGACGCGGAGCTCGAGCTGTGGCGGGTCATCGTACACAGGGTGAAGCTCAACGTGTCGCCCGGCACGTCGTTCCACTGCAACGAGCCCGGCTGGTTCCGCGTCTGCTACGCCAACATGGACGACGACACCATGGAGGTCGCGCTCGACCGGATCCGCCGCTTCGTGCGCCAGCACCAGCACAGCAAGGCCAAGGCCGAGCGCTGGGCGGCCACGCGGCCCCTTCGCCTCAGCTTGCCGCGCCGGGGAGCAACCACCGCTTCGCATCTCGCCATCTCCAGCCCCTTGGCGTTGCTGTCGCCGCAGTCCCCGATGGTCCACGCCAGC SEQ ID: 7Synthase aa ACS2 MAGGSSAEQL LSRIASGDGH GENSSYFDGW KAYDMDPFDL RHNRDGVIQM(ACC2) ACC GLAENQLSLD LIEQWSMEHP EASICTAQGA SQFRRIANFQ DYHGLPEFRESynthase amino AMAKFMGQVR AGKVTFDPDR VVMCGGATGA QDTLAFCLAD PGDAYLVPTPacid sequence YYPAFDRDCC WRSGVKLLPI ECHSSNNFTL TREALVSAYD GARRQGVRVK(A47) from Zea GVLITNPSNP LGTTMDRATL AMLARFATEH RVHLICDEIY AGSVFAKPDFmays (Zm) inbred VSIAEVIERD VPGCNRDLIH IAYSLSKDFG LPGFRVGIVY SYNDDVVACA‘B73’ (= ACC2 pCr RKNSSFGLVS SQTQHFLAKM LSDAEFMARF LAESARRLAA RHDRFVAGLRfragment EVGIACLPGN AGLFSWMDLR GMLRDKTHDA ELELWRVIVH KVKLNVSPGToriginally SFHCNEPGWF RVCHANMDDE TMEVALDRIR RFVRQHQHKA KAERWAATRPisolated from MRLSLPRRGG ATASHLPISS PMALLSPQSP MVHAS inbred ‘Oh43’) SEQID: 8 Synthase aa ACS6 MIADEKPQPQ LLSKKAACNS HGQDSSYFLG WEEYEKNPYDPVANPGGIIQ (ACC6) ACC MGLAENQLSF DLLEAWLEAN PDALGLRRGG ASVFRELALFQDYHGMPAFK Synthase amino NALARFMSEQ RGYRVTFDPS NIVLTAGATS ANEALMFCLADHGDAFLIPT acid sequence PYYPGFDRDL KWRTGAEIVP VHCTSGNGFR LTRAALDDAYRRAQKLRLRV (A65) from Zea KGVLITNPSN PLGTTSPRAD LEMLVDFVAA KGIHLVSDEIYSGTVFADPG mays (Zm) FVSVLEVVAA RAATDDGVVG VGPLSDRVHV VYSLSKDLGLPGFRVGAIYS inbred ‘B73’ (= SNAGVVSAAT KMSSFGLVSS QTQHLLASLL GDRDFTRRYIAENTRRIRER ACC6 pCr REQLAEGLAA VGIECLESNA GLFCWVNMRR LMRSRSFEGEMELWKKVVFE fragment VGLNISPGSS CHCREPGWFR VCFANMSAKT LDVALQRLGAFAEAATAGRR originally VLAPARSISL PVRFSWANRL TPGSAADRKA ER isolated frominbred ‘Oh43’) SEQ ID: 9 Synthase aa ACS7 MAGSSAEQLL SRIAAGDGHGENSSYFDGWK AYDMNPFDLR HNRDGVIQMG (ACC7) ACC LAENQLSLDL IEQWSVDHPEASICTAQGAP MAKFMGQVRG GKVTFDPDRV Synthase amino VMCGGATGAQ DTLAFCLADPGDAYLVPTPY YPAFDRDCCW RSGVKLLPIE acid sequence CHSSNNFTLT REALVSAYDGARRQGVRVRG ILITNPSNPL GTTMDRGTLA (A50) from Zea MLAAFATERR VHLICDEIYAGSVFAKPGFV SIAEVIERGD APGCNRDLVH mays (Zm) IAYSLSKDFG LPGFRVGIVYSYNDDVVACA RKMSSFGLVS SQTQHFLAMM inbred ‘B73’ (= LADAEFMARF LAESARRLAARHDRFVAGLR EVGIACLPGN AGLFSWNDLR ACC7 pCr GMLREKTHDA ELELWRVIVHRVKLNVSPGT SFHCNEPGWF RVCYANMDDD fragment TMEVALDRIR RFVRQHQHSKAKAERWAATR PLRLSLPRRG ATTASHLAIS originally SPLALLSPQS PMVHAS isolatedfrom inbred ‘Oh43’) SEQ ID: 10 CCRA178 R ATGACCATGA TTACGCCAAGCTCTAATACG ACTCACTATA GGGAAAGCTG GTACGCCTGC AGGTACCGGT CCGGAATTCCCGGGTCGACC CACGCGTCCG CAGCAAGCTC ATCCCCTTCA AAACCCTCGG GCAGCCCAGCCAGCTAGTGG TGATCTCTCA GCAGCGCGCC TGAACGTGTG CTCCCTGCTA AACTCTGCGCCTCGGTAGGC AAGGAAAATT AAACCGGTCG TCGTCAGATT AAATGGCCGG TAGCAGCGCGGAGCAGCTCC TCTCCAGGAT CGCCGCCGGC GATGGCCACG GCGAGAACTC GTCCTACTTCGACGGGTGGA AGGCCTACGA CACGAACCCT TTCGACCTGC GCCACAACCG CGACGGCGTCATCCAGATGG GACTCGCCGA GAACCAACTG TCGCTGGACC TGATCGAGCA ATGCAGCGTGGACCACCCCG AGGCGTCCAT CTGCACGGCG CAGGGCGCGC CGCAGTTCCG GAGGATAGCCAACTTCCAGG ACTACCACGG CCTGCCGGAG TTCAGAGAGG CGATGGCCAA GTTCATGGGGCAGGTGAGGG GCGGCAAGGT GACGTTCGAC CCCGACCGCG TCGTCATGTG CGGGGGAGCCACCGGCGCGC AGGACACTCT CGCCTTCTGC CTCGCTGACC CGGGCGACGC CTACCTCGTGCCGACGCCTT ATTACCCAGC TTTCGACCGC GACTGTTGCT GGAGGTCAGG AGTGAAGCTGCTGCCCATCG AATGCCACAG CTCGAACAAC TTCACCCTCA CCAGGGAGGC GCTCGTGTCGGCCTACGACG GCGCGCGGAG GCAGGGCGTC CGCGTCAGGG GCATCCTCAT CACCAACCCCTCCAACCCGC TGGGCACCAC AATGGACCGC GGCACGCTGG CGATGCTCGC CGCGTTCGCCACAGAGCGCC GCGTCCACCT CATCTGCGAC GAGATCTACG CGGGCTCCGT CTTCGCCAAGCCGGGCTTCG TGAGCATCGC CGAGGTCATC GAGCGCGGCG ACGCCCCGGG CTGCAACAGGGACCTCGTCC ACATCGCGTA CAGCCTCTCC AAGGACTTCG GCCTCCCGGG CTTCCGCGTCGGCATCGTCT ACTCCTACAA CGACGACGTG GTGGCCTGCG CGCGCAAGAT GTCCAGCTTCGGCCTCGTCT CGTCGCAGAC GCAGCACTTC CTGGCGATGA TGCTCGCCGA CGCGGAGTTCATGGCACGCT TCCTCGCGGA GAGCGCGCGG CGGCTGGCGG CGCGCCACGA CCGCTTCGTCGCGGGCCTCC GCGAGGTCGG CATCGCGTGC CTGCCGGGCA ACGCGGGCCT CTTCTCGTGGATGGACCTGC GGGGCATGCT CCGGGAGAGG ACGCACGACG CGGAGCTGGA GCTGTGGCGGGTCATCGTAC ACAGGGTGAA GCTCAACGTG TCGCCCGGCA CGTCGTTCCA CTGCAACGAGCCCGGCTGGT TCCGCGTCTG CTACGCCAAC ATGGACGACG ACACCATGGA GGTCGCGCTCGACCGGATCC GCCGCTTCGT GCGCCAGCAC CAGCACAGCA AGGCCAAGGC CGAGCGCTGGGCGGCCACGC GGCCCCTCCG CCTCAGCTTG CCGCGCCGGG GAGCAACCAC CGCTTCGCACCTCGCCATCC CCAGCCCCTT GGCGTTGCTG TCGCCGCAGT CCCCGATGGT CCACGCCAGCTAGCTAGTCA CCGAGCGTTC GGTAAGACTG GCTGTAGGGT GTGCCCTCAC ATAACTGCAAACAAGTGGAC AAAAAATATT AGACAAGACT AATAAAGGGC ATTAGTAGCT AGCTTGACATTACACAGAGA CGTTGCACAG GCGTCAGCAG GCGTCGGCGG TAAGCAGCTA GTCAAGCAGGACGCATTTGT CCTCGATTTT TTCGTGTATA TATGTTCTTT TTTCTGTTTT GCCAAATCGCATGTATGGTT TGGTTTAACG TTAGTACACG GTAGAATAAC GATCGGGTAT GGTAATTTAGACCTCCCGAT CAATTGTTGT TGAAAACCTG TCACGTAACT TCAGGACACA GAAGGCGTAGCTCAAGGGTG AATAAAAGAC CAGTTTACAT ATCAAAAAAA AAAAAAAAAA AAAAAAAAAA SEQID: 11 CCRA178 R MAGSSAEQLL SRIAAGDGHG ENSSYFDGWK AYDTNPFDLR HNRDGVIQMGaa LAENQLSLDL IEQWSVDHPE ASICTAQGAP QFRRIANFQD YHGLPEFREA MAKFMGQVRGGKVTFDPDRV VMCGGATGAQ DTLAFCLADP GDAYLVPTPY YPAFDRDCCW RSGVKLLPIECHSSNNFTLT REALVSAYDG ARRQGVRVRG ILITNPSNPL GTTMDRGTLA MLAAFATERRVHLICDEIYA GSVFAKPGFV SIAEVIERGD APGCNRDLVH IAYSLSKDFG LPGFRVGIVYSYNDDVVACA RKMSSFGLVS SQTQHFLANM LADAEFMARF LAESARRLAA RHDRFVAGLREVGIACLPGN AGLFSWMDLR GMLRERTHDA ELELWRVIVH RVKLNVSPGT SFHCNEPGWFRVCYANMDDD TMEVALDRIR RFVRQHQHSK AKAERWAATR PLRLSLPRRG ATTASHLAIPSPLALLSPQS PMVHAS SEQ ID: 12 ACCF1 ccagatgggcctcgccgagaac (forwardprimer) SEQ ID: 13 ACC1 gttggcgtagcagacgcggaacca (reverse primer)

1. An isolated or recombinant knockout plant cell comprising at least one disruption in at least one endogenous ACC synthase gene, wherein the disruption inhibits expression or activity of at least one ACC synthase protein compared to a corresponding control plant cell lacking the disruption.
 2. The plant cell of claim 1, wherein the at least one endogenous ACC synthase gene comprises a nucleic acid sequence, or complement thereof, comprising at least about 85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), or SEQ ID NO:3 (gACS7).
 3. The plant cell of claim 1, wherein the at least one endogenous ACC synthase gene comprises two or more endogenous ACC synthase genes.
 4. The plant cell of claim 1, wherein the at least one endogenous ACC synthase gene comprises three or more endogenous ACC synthase genes.
 5. The plant cell of claim 1, wherein the at least one disruption results in reduced ethylene production by the knockout plant cell as compared to the control plant cell.
 6. The plant cell of claim 1, wherein the at least one disruption comprises one or more transposons, wherein the one or more transposons are in the at least one endogenous ACC synthase gene.
 7. The plant cell of claim 6, wherein the at least one disruption is a homozygous disruption in the at least one ACC synthase gene.
 8. The plant cell of claim 6, wherein the at least one disruption is a heterozygous disruption in the at least one ACC synthase gene.
 9. The plant cell of claim 1, wherein the at least one disruption comprises one or more point mutations, wherein the one or more point mutations are in the at least one endogenous ACC synthase gene.
 10. The plant cell of claim 1, wherein the at least one disruption is introduced into the knockout plant cell by introducing at least one polynucleotide sequence comprising an ACC synthase nucleic acid sequence, or subsequence thereof, into the knockout plant cell, such that the at least one polynucleotide sequence is linked to a promoter in a sense or antisense orientation, and wherein the at least one polynucleotide sequence comprises at least about 85% sequence identity to SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7), or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a complement thereof. 11-12. (Canceled)
 13. The plant cell of claim 1, wherein the at least one disruption is introduced into the knockout plant cell by introducing at least one polynucleotide sequence comprising one or more subsequences of an ACC synthase nucleic acid sequence configured for RNA silencing or interference. 14-17. (Canceled)
 18. The plant cell of claim 19, wherein the dicot or monocot is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 19. The plant cell of claim 1, wherein the plant cell is from a dicot or monocot.
 20. The plant cell of claim 1, wherein the plant cell is in a plant comprising a staygreen potential phenotype.
 21. The plant cell of claim 1, wherein the plant cell is in a plant comprising a male sterility phenotype.
 22. A plant regenerated from the plant cell of claim
 1. 23. A knockout plant comprising a staygreen potential phenotype, the staygreen potential phenotype resulting from a disruption in at least one endogenous ACC synthase gene, wherein the disruption comprises one or more transposons or one or more point mutations, and wherein the disruption inhibits expression or activity of at least one ACC synthase protein compared to a corresponding control plant.
 24. The knockout plant of claim 23, wherein the at least one endogenous ACC synthase gene comprises a nucleic acid sequence, or complement thereof, comprising at least about 85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), or SEQ ID NO:3 (gACS7), or a complement thereof.
 25. The knockout plant of claim 23, wherein the staygreen potential phenotype of the transgenic plant comprises increased drought resistance, increased time for maintaining a photosynthetically active plant, or delayed leaf senescence, compared to the corresponding control plant.
 26. The knockout plant of claim 23, wherein the knockout plant is a hybrid plant.
 27. The knockout plant of claim 23, wherein the knockout plant is a dicot or monocot.
 28. The knockout plant of claim 27, wherein the knockout plant is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 29. A transgenic knockout plant comprising a staygreen potential phenotype, said staygreen potential phenotype resulting from at least one introduced transgene which inhibits ethylene synthesis, wherein said at least one introduced transgene comprises a nucleic acid sequence encoding at least one ACC synthase, which nucleic acid sequence comprises at least about 85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a complement thereof, and is in a configuration that modifies a level of expression or activity of the at least one ACC synthase.
 30. The transgenic knockout plant of claim 29, wherein the configuration comprises an antisense, sense or RNA silencing or interference configuration.
 31. The transgenic knockout plant of claim 29, wherein the transgene comprises a tissue-specific promoter or an inducible promoter.
 32. The transgenic knockout plant of claim 29, wherein the staygreen potential phenotype of the transgenic plant comprises increased drought resistance, increased time for maintaining a photosynthetically active plant, or delayed leaf senescence, compared to a corresponding control plant.
 33. The transgenic knockout plant of claim 29, wherein the plant is a dicot or monocot.
 34. The transgenic knockout plant of claim 33, wherein the plant is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 35. A transgenic plant comprising a staygreen potential phenotype, said staygreen potential phenotype resulting from at least one introduced transgene which inhibits ethylene synthesis, wherein said at least one introduced transgene comprises a nucleic acid sequence encoding a subsequence of at least one ACC synthase, which at least one ACC synthase comprises at least about 85% sequence identity to SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO.:9 (pACS7), or SEQ ID NO:11 (pCCRA178R), is in an RNA silencing or interference configuration, and modifies a level of expression or activity of the at least one ACC synthase.
 36. The transgenic plant of claim 35, wherein the staygreen potential phenotype of the transgenic plant comprises increased drought resistance, increased time for maintaining a photosynthetically active plant, or delayed leaf senescence, compared to a corresponding control plant.
 37. The transgenic plant of claim 35, wherein the plant is a dicot or monocot.
 38. The transgenic plant of claim 37, wherein the plant is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 39. An isolated or recombinant polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide, or a complement thereof, comprising at least about 85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7), or SEQ ID NO:10 (CCRA178R), or a subsequence thereof; (b) a polynucleotide, or a complement thereof, encoding a polypeptide sequence of SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO.:9 (pACS7), or SEQ ID NO:11 (pCCRA178R), or a subsequence thereof, or a conservative variation thereof; and, (c) a polynucleotide, or a complement thereof, that hybridizes under stringent conditions over substantially the entire length of a polynucleotide subsequence comprising at least 100 contiguous nucleotides of SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7), or SEQ ID NO:10 (CCRA178R).
 40. The isolated or recombinant polynucleotide of claim 39, wherein the polynucleotide inhibits ethylene production when expressed in a plant.
 41. An expression cassette comprising a promoter operably linked to the isolated or recombinant polynucleotide of claim
 39. 42. The expression cassette of claim 41, wherein the promoter is a constitutive promoter.
 43. The expression cassette of claim 41, wherein the promoter is an inducible promoter.
 44. The expression cassette of claim 41, wherein the promoter is a tissue-specific promoter.
 45. The expression cassette of claim 44, wherein the tissue-specific promoter is a leaf-specific promoter or an anther-specific promoter.
 46. The expression cassette of claim 41, wherein the polynucleotide is linked to the promoter in an antisense orientation.
 47. The expression cassette of claim 41, wherein the polynucleotide is linked to the promoter in a sense orientation.
 48. The expression cassette of claim 41, wherein the polynucleotide is in an RNA silencing or interference configuration.
 49. A vector comprising a promoter operably linked to the isolated or recombinant polynucleotide of claim
 39. 50. The vector of claim 49, wherein the vector is a viral vector.
 51. A knockout plant comprising a male sterility phenotype, the male sterility phenotype resulting from at least one disruption in at least one endogenous ACC synthase gene, the disruption inhibiting expression or activity of at least one ACC synthase protein compared to a corresponding control plant.
 52. The knockout plant of claim 51, wherein the at least one disruption results in reduced ethylene production by the knockout plant as compared to the control plant.
 53. The knockout plant of claim 51, wherein the at least one disruption comprises one or more transposons, wherein the one or more transposons are in the at least one endogenous ACC synthase gene.
 54. The knockout plant of claim 51, wherein the at least one disruption comprises one or more point mutations, wherein the one or more point mutations are in the at least one endogenous ACC synthase gene.
 55. The knockout plant of claim 51, wherein the at least one disruption is introduced into the knockout plant by introducing at least one polynucleotide sequence comprising one or more subsequences of an ACC synthase nucleic acid sequence configured for RNA silencing or interference.
 56. The knockout plant of claim 51, wherein the male sterility phenotype comprises reduced pollen shedding by the knockout plant as compared to the control plant.
 57. A transgenic knockout plant comprising a male sterility phenotype, said male sterility phenotype resulting from at least one introduced transgene which inhibits ethylene synthesis, wherein said at least one introduced transgene comprises a nucleic acid sequence encoding at least one ACC synthase, which nucleic acid sequence comprises at least about 85% sequence identity to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cACS7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a complement thereof, and is in a configuration that modifies a level of expression or activity of the at least one ACC synthase.
 58. The transgenic knockout plant of claim 57, wherein the configuration comprises an antisense, sense or RNA silencing or interference configuration.
 59. The transgenic knockout plant of claim 57, wherein the transgene comprises a tissue-specific promoter or an inducible promoter.
 60. A method of inhibiting ethylene production in a plant, the method comprising: inactivating one or more ACC synthase genes in the plant, wherein the one or more ACC synthase genes encode one or more ACC synthases, wherein at least one of the one or more ACC synthases comprises at least about 85% identity to SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO:9 (pAC7) or SEQ ID NO:11 (pCCRA178R).
 61. The method of claim 60, wherein at least one of the one or more ACC synthase genes is at least about 85% identical to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6) or SEQ ID NO:3 (gAC7), or a complement thereof.
 62. The method of claim 60, wherein the inactivating step comprises introducing one or more mutations into an ACC synthase gene sequence, wherein the one or more mutations in the ACC synthase gene sequence comprise one or more transposons, thereby inactivating the one or more ACC synthase genes compared to a corresponding control plant.
 63. The method of claim 62, wherein the one or more mutations comprise a homozygous disruption in the one or more ACC synthase genes.
 64. The method of claim 62, wherein the one or more mutations comprise a heterozygous disruption in the one or more ACC synthase genes.
 65. The method of claim 62, wherein the one or more mutations are introduced by a sexual cross.
 66. The method of claim 60, wherein the inactivating step comprises introducing one or more mutations into an ACC synthase gene sequence, wherein the one or more mutations in the ACC synthase gene sequence comprise one or more point mutations, thereby inactivating the one or more ACC synthase genes compared to a corresponding control plant.
 67. The method of claim 60, wherein the inactivating step comprises: (a) introducing into the plant at least one polynucleotide sequence, wherein the at least one polynucleotide sequence comprises a nucleic acid encoding one or more ACC synthases, or a subsequence thereof, and a promoter, which promoter functions in plants to produce an RNA sequence; and, (b) expressing the at least one polynucleotide sequence, thereby inactivating the one or more ACC synthase genes compared to a corresponding control plant.
 68. The method of claim 67, wherein the at least one polynucleotide sequence is introduced by electroporation, micro-projectile bombardment or Agrobacterium-mediated transfer.
 69. The method of claim 67, wherein the polynucleotide is linked to the promoter in a sense orientation.
 70. The method of claim 67, wherein the polynucleotide is linked to the promoter in an antisense orientation.
 71. The method of claim 67, wherein the polynucleotide is configured for RNA silencing or interference.
 72. The method of claim 67, wherein the promoter is a tissue-specific promoter or an inducible promoter.
 73. The method of claim 60, wherein the plant is a dicot or monocot.
 74. The plant of claim 73, wherein the plant is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 75. A plant produced by the method of claim
 60. 76. A method for modulating staygreen potential in a plant, the method comprising: a) selecting at least one ACC synthase gene to mutate, thereby providing at least one desired ACC synthase gene; b) introducing a mutant form of the at least one desired ACC synthase gene into the plant; and, c) expressing the mutant form, thereby modulating staygreen potential in the plant.
 77. The method of claim 76, wherein the staygreen potential of the plant comprises: (a) a reduction in the production of at least one ACC synthase specific mRNA; (b) a reduction in the production of an ACC synthase; (c) a reduction in the production of ethylene; (d) a delay in leaf senescence; (e) an increase in drought resistance; (f) an increased time in maintaining photosynthetic activity; (g) an increased transpiration; (h) an increased stomatal conductance; (i) an increased CO2 assimilation; (j) an increased time in maintaining CO2 assimilation; or, (k) any combination of (a)-(j); compared to a corresponding control plant.
 78. The method of claim 76, wherein the selecting the at least one ACC synthase gene comprises determining a degree of staygreen potential desired.
 79. The method of claim 78, wherein the degree of staygreen potential desired is weak, moderate or strong.
 80. The method of claim 76, wherein the ACC synthase gene encodes an ACC synthase selected from the group consisting of: SEQ ID NO:7 (pACS2), SEQ ID NO:8 (pACS6), SEQ ID NO:9 (pAC7) and SEQ ID NO:11 (pCCRA178R).
 81. The method of claim 76, wherein the mutant form comprises a heterozygous mutation in the at least one ACC synthase gene.
 82. The method of claim 76, wherein the mutant form comprises a homozygous mutation in the at least one ACC synthase gene.
 83. The method of claim 76, wherein the mutant form comprises a subsequence of the at least one desired ACC synthase gene in an antisense, sense or RNA silencing or interference configuration.
 84. The method of claim 76, wherein the mutant form is introduced by Agrobacterium-mediated transfer, electroporation, micro-projectile bombardment, or a sexual cross.
 85. The method of claim 76, wherein the plant is a dicot or monocot.
 86. The plant of claim 85, wherein the plant is Zea mays, wheat, rice, sorghum, barley, oat, lawn grass, rye, soybean, Brassica, or sunflower.
 87. A plant produced by the method of claim
 76. 88. A kit for modulating staygreen potential in a plant, the kit comprising: at least one polynucleotide sequence comprising a nucleic acid sequence, wherein the nucleic acid sequence is at least about 85% identical to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cAC7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a complement thereof.
 89. The kit of claim 88, wherein said kit further comprises instructional materials for the use of the at least one polynucleotide sequence to control staygreen potential in a plant.
 90. A kit for modulating male sterility in a plant, the kit comprising: at least one polynucleotide sequence comprising a nucleic acid sequence, wherein the nucleic acid sequence is at least about 85% identical to SEQ ID NO:1 (gACS2), SEQ ID NO:2 (gACS6), SEQ ID NO:3 (gACS7), SEQ ID NO:4 (cACS2), SEQ ID NO:5 (cACS6), SEQ ID NO:6 (cAC7) or SEQ ID NO:10 (CCRA178R), or a subsequence thereof, or a complement thereof.
 91. The kit of claim 90, wherein said kit further comprises instructional materials for the use of the at least one polynucleotide sequence to control male sterility in a plant. 